Heterodimeric fc-fused cytokine and pharmaceutical composition comprising the same

ABSTRACT

The present invention relates to a heterodimeric Fc-fused protein comprising a first Fc region and a second Fc region of an immunoglobulin Fc pair and a physiologically active protein composed of two or more different subunits, wherein one or more subunits of the physiologically active protein are linked separately to one or more ends of the N-terminus or C-terminus of the first Fc region and/or the second Fc region, and CH3 domains of the first Fc region and the second Fc region are mutated so as to promote the heterodimeric Fc formation. Moreover, the present invention relates to a pharmaceutical composition comprising the heterodimeric Fc-fused protein. The heterodimeric Fc-fused protein according to the present invention has an advantage in that it can retain the activity of a naturally occurring physiologically active protein whose two or more different subunits exhibit physiological activity by forming a protein complex, because the physiologically active protein can be linked to an immunoglobulin heterodimeric Fc such that the naturally occurring form and structure of the fused protein thereof can be maintained. When the heterodimeric Fc-fused protein according to the present invention is used, there is an advantage in that the in vivo half-life of the physiologically active protein contained in the heterodimeric Fc-fused protein can be significantly increased due to the Fc-mediated long half-life such that various physiological activities thereof in vivo can be long-lasting.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional patent application under 35 U.S.C. §120 of U.S. patent application Ser. No. 16/323,839, filed on Feb. 7,2019 (now U.S. Pat. No. 10,696,722), which application is a U.S.national stage application under 35 U.S.C. § 371 of InternationalApplication No. PCT/KR2017/008676, filed on Aug. 10, 2017, which claimspriority to and the benefit of Korean Patent Application No.10-2016-0101823, filed on Aug. 10, 2016 and Korean Patent ApplicationNo. 10-2017-0101594, filed on Aug. 10, 2017, each of which isincorporated by reference herein in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 26, 2020, isnamed DFY-300D1_SL.txt and is 25,787 bytes in size.

TECHNICAL FIELD

The present invention relates to a heterodimeric Fc-fused proteincomprising a first Fc region and a second Fc region of an immunoglobulinFc pair and a physiologically active protein, wherein one or moresubunits of the physiologically active protein are linked to one or moreends of the N-terminus or C-terminus of the first Fc region and/or thesecond Fc region, and CH3 domains of the first Fc region and the secondFc region are mutated so as to promote Fc heterodimer formation, and apharmaceutical composition comprising the heterodimeric Fc-fusedprotein.

The heterodimeric Fc-fused protein according to the present inventionhas an advantage in that it can retain the activity of a naturallyoccurring physiologically active protein, which is composed of two ormore different subunit proteins and thereby exhibit the intactbiological activity by forming a assembled protein, because each subunitof the protein can be separately fused to each chain of heterodimeric Fcof immunoglobulin such that the fused protein can maintain the naturallyoccurring form and structure to the highest possible degree.

When the heterodimeric Fc-fused protein according to the presentinvention is used, there is an advantage in that the in vivo half-lifeof the physiologically active protein contained in the heterodimericFc-fused protein can be significantly increased due to the Fc-mediatedlong half-life such that the physiological activities thereof in vivocan be long-lasting.

In addition, the heterodimeric Fc-fused protein according to the presentinvention has a structure in which one or more subunits of thephysiologically active protein are fused to the N-terminus or C-terminusof an immunoglobulin heterodimeric Fc, and the heterodimeric Fc-fusedprotein is easily purified after its expression, compared to a wild-typeFc-based fusion protein.

BACKGROUND ART

Naturally occurring human antibodies (immunoglobulin G (IgG), IgM, IgD,IgE, and IgA) are each present as an assembly of two heavy chains havingthe same amino acid sequence and two light chains having the samesequence. In this regard, homodimerization between the two identicalheavy chains is induced by the non-covalent interactions between theconstant region terminal domains (CH3 domains in IgG, IgD and IgA, CH4domains in IgM, and CH2 and CH4 domains in IgE) and the disulfide bondbetween hinge domains.

Antibody heterodimeric Fc technology is a technology that makesheterodimeric fragment crystallizable (Fc) of immunoglobulin heavy chainconstant regions by modifications to the CH3 domain interface, withdifferent mutations on each domain such that the engineered Fcfragments, carrying the CH3 variant pair, preferentially form Fcheterodimers in naturally occurring antibodies (IgG, IgM, IgA, IgD, andIgE) rather than the Fc homodimers. More specifically, it is atechnology that induces mutations in two different CH3 domains of Fc bygenetic engineering, such that the two Fc fragments form a heterodimerwith minimal sequence variations while they have tertiary structuresvery similar to those of naturally occurring antibodies (U.S. Pat. No.7,695,936; and Korean Patent No. 1,522,954). The heterodimeric Fctechnology is a platform technology for making bispecific antibodies,and CH3 domain mutants that induce Fc heterodimer formation known so farwere mostly generated by introducing an asymmetric mutation pair intothe CH3 domain interface by the structure-based rational design ofantibody (Spreter Von Kreudenstein et al., 2014). Pioneering studiesinclude knob-into-hole technology (Ridgway et al., 1996) from Genentech,and many multinational pharmaceutical companies, including Zymeworks(ZW1; Von Kreudenstein et al., 2013), Xencor (HA-TF; Moore G L et al.,2011) and EMD Serono (SEEDbody; Davis J H et al., 2010), have developedand reported the platform technology.

Above all, the A107 variant used in the present invention is ahigh-yield Fc heterodimer screened from a human antibody heterodimericFc library constructed using a yeast cell surface display system, and isa heterodimeric Fc variant which promotes the heterodimeric formation byinducing mutations at charged amino acids to form stericallycomplementary hydrophobic interactions (K409W_(CH3A)-D399V/F405T_(CH3B))and forming hydrogen bonds (K370E_(CH3A)-E357N_(CH3B)), while retaininghydrophobic core integrity at the CH3 domain interface (Choi et al.2016; Korean Patent Application No. 2015-0142181).

Heterodimeric Fc variants reported so far, including the A107 variant,are all based on IgG1 occupying the largest proportion of human antibodyisotypes, and variants of isotypes (IgG2, IgG3, IgG4, IgA, IgM, and IgE)other than IgG1 have not been reported yet.

This is because therapeutic antibodies that are being marketed underapproval of the U.S. Food and Drug Administration (FDA) mostly adopt theIgG1 isotype (Irani et al. 2015). In recent years, for immune-modulatingantibodies or receptor agonist fusion proteins that do not need to havegreat antibody effector functions such as antibody-dependent cellularcytotoxicity (ADCC) or complement-dependent cellular cytotoxicity (CDC),the development of therapeutic proteins based on IgG2 or IgG4 whoseeffector functions are significantly lower than those of IgG1 have beenmade.

Meanwhile, physiologically active proteins mostly have small sizes, andthus have the disadvantage of having a short in vivo half-life. In orderto solve this disadvantage, there has been an attempt to conjugate PEG(polyethylene glycol) or the like, or fusion to an antibody Fc(crystallizable fragment) region. However, it has not yet been possibleto develop physiologically active proteins whose activity is efficientlyand sufficiently maintained for a long period of time.

In particular, for proteins composed of two or more different subunits,wherein the two or more different subunits form a protein complex toexhibit physiological activity, it has never been possible to developFc-fused proteins which are formed to have naturally occurring originalprotein complex structures with wild type Fc because wild type Fc-fusedprotein forms homodimer due to the homodimeric nature of Fc. Thus, wildtype Fc is not suitable for Fc fusion for heterodimeric orheterooligomeric proteins to properly exhibit the activity of theoriginal proteins and sufficiently maintain their activity for a longperiod of time.

Under this technical background, the present inventors have constructedheterodimer variants comprising Fc regions derived not only from IgG1,but also from other isotype antibodies such as IgG2, IgG3 and IgG4,which were previously not reported, and have used these heterodimervariants to develop a novel therapeutic fusion protein in the form of aheterodimeric Fc-fused protein wherein one or more subunits of aprotein, which is composed of two or more different subunits and inwhich two or more subunits exhibit physiological activity by forming aprotein complex, are genetically fused to the terminus of the Fc region,thereby completing the present invention.

In particular, in the present invention, preferably, interleukin-12(IL-12) can be used as the protein which is composed of two differentsubunits, p35 and p40, wherein the two subunits exhibit physiologicalactivity by forming the IL-12 protein.

IL-12 can directly kill tumors by increasing the activity of immunecells such as cytotoxic T lymphocytes (CTLs) or natural killer cells(NKs) among immune cells, or can inhibit tumorigenesis by activatingimmune responses through secretion of pro-inflammatory cytokines such asinterferon-gamma (IFN-y) in tumor microenvironments where the immuneresponses are inhibited. Thus, IL-12 has been much studied as ananti-cancer cytokine (Lasek et al., 2014). However, in the developmentof therapeutic methods using IL-12, the short half-life of the cytokineitself necessitates frequent administration which can lead to toxicity.For this reason, studies have been conducted to fuse IL-12 with anantibody or Fc in order to use it as long-acting IL-12 (Tugues et al.,2015). However, in these studies, a problem arises in that, due to thefusion of a wild-type

Fc-based antibody that forms a homodimer by the interaction between CH3domains, the fused IL12 protein is bivalent, unlike an endogenousmonovalent form of IL-12, and for this reason, the wild type Fc-basedantibody fused IL-12 shows poor physiological activity than endogenousIL-12, or unwanted localization appears due to avidity-driven increasedbinding of IL-12 to immune cells (Tzeng et al., 2015; Dumont et al.,2006).

Therefore, in an effort to make a monovalent fusion protein using awild-type antibody or an Fc region, as shown in FIGS. 1(A) to 1(C),there has been used a method of constructing a fusion protein through astrategy such as fusing a selective tag for additional purification onlyto the C-terminus of a single Fc region or fusing an Fc region and aprotein to each other after separately purifying them with high purity.However, this method is not only very costly to produce a large amountof protein, but also requires research to optimize an additionalpurification process.

However, the use of a heterodimeric Fc-fused protein according to thepresent invention makes it possible to easily produce a monovalentheterodimeric Fc-fused protein as shown in FIGS. 2A-2C without needingto optimize an additional purification process.

DISCLOSURE OF INVENTION Technical Problem

It is an object of the present invention to provide a novelheterodimeric Fc-fused protein, the protein of which is composed of one,two, or more different subunits and thereby exhibits the intactbiological activity by forming the assembled protein, and thus canmaintain the natural physiological activity of the fused protein thereofin vivo for a long period of time.

In particular, the heterodimeric Fc-fused protein according to thepresent invention is formed such that it can retain the activity of anaturally occurring physiologically active protein, in which two or moresubunits assemble together to form a protein to exhibit physiologicalactivity, such that the fused protein can maintain the naturallyoccurring form and structure to the highest possible degree.

Further, the heterodimeric Fc-fused protein according to the presentinvention has an advantage in that the in vivo half-life of thephysiologically active protein contained in the heterodimeric Fc-fusedprotein can be significantly increased due to the Fc-mediated longhalf-life such that the physiological activities thereof in vivo can belong-lasting.

Another object of the present invention is to provide a pharmaceuticalcomposition comprising the above-described heterodimeric Fc-fusedprotein, and a composition and a therapeutic method for treatingdiseases, particularly cancer, using the same.

Technical Solution

To achieve the above object, the present invention provides aheterodimeric Fc-fused protein comprising a first Fc region and a secondFc region of an immunoglobulin Fc pair and a physiologically activeprotein, wherein the physiologically active protein is composed of twoor more different subunits, wherein the two or more different subunitsexhibit physiological activity by forming a protein complex, wherein thesubunits of a physiologically active protein are linked or geneticallyfused to one or more ends of the N-terminus or C-terminus of the firstFc region and/or the second Fc region, wherein the CH3 domains of thefirst Fc region and the second Fc region are mutated so as to promote Fcheterodimer formation.

The present invention also provides a pharmaceutical compositioncomprising the above-described heterodimeric Fc-fused protein, and acomposition and a therapeutic method for treating diseases, particularlycancer, using the same.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1C illustrate conventional strategies for obtainingmonomeric and heterodimeric fusion proteins using wild-type Fc of humanIgG antibody. FIG. 1A is an illustrative diagram of a wild-type Fc-basedEpo-Fc dimer and a wild-type Fc-based Epo-Fc monomer. FIG. 1B is anillustrative diagram of an aglycosylated Fc-fused GLP-1/GCG monomericpeptide, generated by the LAPScovery technology. FIG. 1C is anillustrative diagram of a wild-type Fc-based Fc-FSH tandem homodimer anda wild-type Fc-based Fc-FSH heterodimer. Epo, erythropoietin; GLP-1/GCG,glucagon-like peptide-1/glucagon; FSH, Follicle-stimulating hormone.

FIG. 1D is an illustrative diagram of an exemplary antibody-cytokine(immunocytokine) constructed by fusing a monomeric cytokine (IL2) to anIgG type antibody comprising a knob-into-hole (KiH) heterodimeric Fcvariant according to previous literature.

FIG. 2A illustrates monomeric fusion protein forms which may beconstructed using a heterodimeric Fc. The Fc-fused monomer can easily begenerated by the fusion of monomeric protein to the N- or C-terminus ofone heterodimeric Fc chain. FIG. 2B illustrates heterodimeric fusionprotein forms which may be constructed using a heterodimeric Fc.Potential use of heterodimeric Fc for the generation of Fc-fusedmonomeric or heterodimeric proteins to present the fusion partner in itsnaturally occurring form.

FIG. 2C illustrates a fusion protein formed by fusing a heterodimer toan IgG type human antibody comprising a heterodimeric Fc. The Fc-fusedheterodimer can be generated by separate fusion of the two subunits ofheterodimeric proteins to each chain of the heterodimeric Fc at the N-or C-terminus.

FIG. 3 shows the sequence alignment of CH3 domain of human IgG isotypeantibodies (hIgG1, hIgG2, hIgG3, hIgG4) with highlights of the mutatedresidues in A107 heterodimeric Fc variant(K370E/K409W_(CH3A)-E357N/D399V/F405T_(CH3B)).

FIG. 4 shows the results of performing structural modeling ofheterodimeric Fc variants for each isotype by use of sequences havinginduced mutations at the positions selected in FIG. 3 and analyzing theresulting modeling structures comparatively with wild-type IgG1-basedA107 variants.

FIG. 5 is a schematic view of a vector for expressing a heterodimeric Fcfor each isotype, constructed by sequence and structure analysis, inanimal cells. The heterodimeric Fc variant for each isotype, whichcomprises a mutated hinge region, was cloned into the vector by use ofrestriction enzymes (Notl/HindIII).

FIG. 6 schematically shows a scFv-Fc_(CS3A)/Fc_(CH3B) expression systemfor evaluating the ability of heterodimeric Fc variants to form aheterodimer, by the dimer size difference between expressed proteins.

FIG. 7 is a schematic view for cloning scFv-Fc fused to a single-chainvariable fragment (scFv), constructed to evaluate the heterodimerizationformation yield of an antibody Fc by a CH3 mutant pairs as shown in FIG.6, into a pcDNA3.1 vector which is an animal cell expression vector.

FIG. 8 shows the results of co-transfecting CH3 mutant pairs-introducedanimal cell expression vectors, constructed according to the expressionsystems shown in FIGS. 5 and 7, into HEK293F cells in order to evaluatethe heterodimerization formation as shown in FIG. 6, transientlyexpressing and purifying the vectors, and then separating 5 μg ofprotein on SDS-PAGE under non-reducing conditions in order to evaluatethe heterodimerization formation, and analyzing the protein according tosize and combination by Coomassie blue staining. As a negative control,a wild-type Fc with wild-type CH3 was used.

FIG. 9 shows the results of separating protein by SDS-PAGE according tothe method shown in FIG. 8, and then performing Western blotting usinganti-human IgG-AP conjugated antibody.

FIG. 10A is a schematic view showing the form of endogenous IL-12cytokine to which Fc was not fused and which is used as a control in thepresent invention.

FIG. 10B is a schematic view showing the form of a bi-IL-12-Fc fusionprotein which was obtained by fusing IL-12 cytokine to wild-type IgG4 Fcby an amino acid linker and which is used as a comparative example inthe present invention.

FIG. 10C is a schematic view showing the form of a mono-IL-12-Fc fusionprotein obtained by fusing IL-12 cytokine to an IgG4-based γ4-A107variant among heterodimeric Fc variants for each isotype according tothe present invention.

FIG. 11A shows a schematic view of a vector for expressing and purifyingan IL-12 p40 fusion protein of an example of the present invention (FIG.10C) in animal cells. FIG. 11B shows a schematic view of a vector forexpressing and purifying an IL-12 p35 fusion protein of an example ofthe present invention (FIG. 10C) in animal cells.

FIG. 12 is a schematic view of a vector for expressing and purifying afusion protein of an example of the present invention (FIG. 10B) inanimal cells.

FIG. 13 shows the results of co-transfecting the animal cell expressionvectors of FIGS. 11A and 11B, constructed using human and mouseinterleukin genes, into HEK293F cells, transiently expressing andpurifying the genes, and then separating 5 μg of protein on SDS-PAGEunder non-reducing conditions, and analyzing the protein according tosize and combination by Coomassie blue staining.

FIG. 14 shows the results of analyzing the fusion proteins of FIG. 13 bysize-exclusion chromatography (SEC).

FIG. 15A are flow-cytometry histograms showing the results of FACSanalysis performed to analyze the binding affinities of mono-hIL-12-Fcand wild-type bi-hIL-12-Fc on normal PBMCs having no IL-12 receptor.FIG. 15B are flow-cytometry histograms showing the results of FACSanalysis performed to analyze the binding affinities of mono-hIL-12-Fcand wild-type bi-hIL-12-Fc on phytohaemagglutinin (PHA)-activated PBMCsin which the IL-12 receptor was induced by treatment with the mitogenPHA.

FIGS. 16A-16D show the results of a WST-1 cell proliferation assayperformed using cells from donor 1 (FIG. 16A), donor 2 (FIG. 16B), donor3 (FIG. 16C), or donor 4 (FIG. 16D) to measure the effect of variousconcentrations of Fc (A107), recombinant human IL-12 (rhIL-12),bi-hIL-12-Fc and mono-hIL-12-Fc on the proliferation of PHA-activatedPBMCs in which the IL-12 receptor was induced by treatment with themitogen PHA.

FIGS. 17A-17D show the results of an ELISA performed using cells fromdonor 1 (FIG. 17A), donor 2 (FIG. 17B), donor 3 (FIG. 17C), or donor 4(FIG. 17D) to measure the concentration of IFN-γ in culture supernatantsobtained as shown in FIG. 16.

FIG. 18A are histograms showing the results of flow cytometry analysisperformed to measure the binding affinities of mono-mIL-12-Fc andbi-mIL-12-Fc on normal PBMCs having no IL-12 receptor. FIG. 18B arehistograms showing the results of flow cytometry analysis performed tomeasure the binding affinities of mono-mIL-12-Fc and bi-mIL-12-Fc onPHA-activated PBMCs in which the IL-12 receptor was induced by treatmentwith the mitogen PHA, because mIL-12 cross-reacts with human IL-12R onactivated human T cells and NK cells.

FIGS. 19A and 19B show the results of a WST-1 cell proliferation assayperformed using cells from donor 1 (FIG. 19A) or donor 2 (FIG. 19B) tomeasure the effect of various concentrations of Fc (A107), recombinantmouse IL-12(rmIL-12), bi-mIL-12-Fc and mono-mIL-12-Fc on theproliferation of PHA-activated PBMCs in which the IL-12 receptor wasinduced by treatment with the mitogen PHA.

FIG. 20A is a graph showing the changes of tumor volume in Balb/c micetransplanted with CT26^(HER2)/Neu tumors during the intraperitonealadministration of Fc (A107), rmIL-12, bi-mIL-12-Fc and mono-mIL-12-Fc.FIG. 20B shows pictures of the tumor-bearing mice after sacrifice at theend of administration. Injection of mIL12-Fc proteins was initiated 11days after tumor cell inoculation when the tumor volume reached 100 mm3.

FIG. 20C is a graph showing the changes of mouse body weight measured atindicated time points in the experimental procedure shown in FIG. 20A.

FIGS. 21A-21E are graphs showing the results of measuring mouse tumorvolume changes measured while intraperitoneally administering variousconcentrations (0.1 μg (FIG. 21A), 0.25 μg (FIG. 21B), 0.5 μg (FIG.21C), 1 μg (FIG. 21D), or 2 μg (FIG. 21E)) of bi-mIL-12-Fc andmono-mIL-12-Fc, twice a week, when the tumor volume in Balb/c micetransplanted with CT26^(HER2/Neu) reached 300 mm³.

FIG. 21F-21L are graphs showing the changes of individual mouse tumorvolume treated with various concentrations (0 μg (FIG. 21F), 0.1 μg(FIG. 21G), 0.25 μg (FIG. 21H), 0.5 μg (FIG. 21I), 1 μg (FIG. 21J), 2 μg(FIG. 21K), or 4 μg (FIG. 21L)) of mIL12-Fc proteins at indicated timepoints in the experimental procedure shown in FIGS. 21A-21E.

FIG. 21M shows pictures of tumor-bearing mice 3 days after the lastadministration of mono-mIL-12-Fc as described in Example 14.

FIG. 21N shows pictures of tumor-bearing mice 3 days after the lastadministration of bi-mIL-12-Fc as described in Example 14.

FIG. 21O is a graph showing the changes in body weight of mono-mIL12-Fc-administered mice measured at indicated time points in theexperimental procedure shown in FIGS. 21A-21E.

FIG. 21P is a graph showing the changes in body weight of bi-mIL12-Fc-administered mice measured at indicated time points in theexperimental procedure shown in FIGS. 21A-21E.

FIG. 21Q is a graph showing the results of measuring alanineaminotransferase (ALT) (which is a hepatotoxicity marker) in the bloodwhich was collected from mouse facial veins 1 day after the lastadministration in FIGS. 21A-21E.

FIGS. 22A-22C are graphs showing the results of measuring increases inthe number of CD4⁺ T cells (FIG. 22A), CD8⁺ T cells (FIG. 22B), and NKcells (FIG. 22C) in the spleens of mice sacrificed 3 days after the lastadministration in FIGS. 21A-21E.

FIGS. 22D-22F are graphs showing the number of total immune cells (FIG.22D), CD4⁺ T cells (FIG. 22E) and CD8⁺ T cells (FIG. 22F) thatinfiltrated the tumor in mice sacrificed 3 days after the thirdadministration in FIGS. 21A-21E.

FIGS. 23A-23B show the results of an ELISA performed to measure theserum levels of IFN-γ in CT26^(HER2/neu) tumor bearing mice treated withmIL-12-Fc proteins. FIG. 23A shows the results of an ELISA performed tomeasure the serum levels of IFN-γ in CT26^(HER2/neu) tumor bearing micetreated with mono-mIL-12-Fc. FIG. 23B shows the results of an ELISAperformed to measure the serum levels of IFN-γ in CT26^(HER2/neu) tumorbearing mice treated with bi-mIL-12-Fc. Mouse serum was separated afterclotting blood collected from mouse facial veins at 24 hours after thelast administration in FIGS. 21A-21E.

FIG. 23C is a graph showing the results of an ELISA performed to measurethe concentration of IFN-γ in serum separated from blood collected frommouse facial veins on 1, 3 and 5 days after intraperitoneallyadministering bi-mIL-12-Fc and mono-mIL-12-Fc at a concentrationequimolar to 1 μg rmIL-12 when the tumor volume in Balb/c micetransplanted with CT26^(HER2/Neu) cancer cells reached 300 mm³.

FIG. 23D is a graph showing the results of measuring the cytotoxiceffect of cytotoxic T cells, isolated from the spleen of mice sacrificed3 days after the last administration in FIGS. 21A-21E, againstCT26^(HERS/Neu) cancer cells.

FIGS. 23E-23F are flow cytometry dot plots showing the cytotoxicactivity of splenic CD8⁺ T cells isolated from CT26-HER2/neutumor-bearing mice treated with mIL-12-Fc proteins, analyzed 3 daysafter the third administration in FIGS. 21A-21E, followed by 4 h ofculture with CT26^(HERS/Neu) cancer cells expressing tumor antigen and4T1 cells not expressing tumor antigen.

FIG. 23G are graphs showing the cytotoxic activity of splenic CD8⁺ Tcells isolated from CT26-HER2/neu tumor-bearing mice treated withmIL-12-Fc proteins, analyzed 3 days after the third administration inFIGS. 21A-21E, followed by 4 h of culture with CT26^(HERS/Neu) cancercells expressing tumor antigen and 4T1 cells not expressing tumorantigen.

FIG. 23H is a graph showing the results of measuring the cytotoxiceffect of natural killer cells, isolated from the spleen of micesacrificed 3 days after the third administration in FIGS. 21A-21E,against CT26^(HERS/Neu) cancer cells.

FIG. 24A is a graph showing the results of measuring the number of CD8⁺effector T cells isolated from in the spleen isolated from tumor-bearingmice sacrificed 3 days after the last administration in FIGS. 21A-21E.

FIG. 24B is a graph showing the results of measuring the number of CD8⁺effector memory T cells in the spleen isolated from tumor-bearing micesacrificed 3 days after the last administration in FIGS. 21A-21E.

FIG. 24C is a graph showing the results of measuring the number of CD8⁺central memory T cells in the spleen isolated from tumor-bearing micesacrificed 3 days after the last administration in FIGS. 21A-21E.

FIG. 24D shows the results obtained by re-transplanting CT26^(HER2/Neu)cancer cells into survived Balb/c mice 120 days after administration of1 μg mono-IL-12-Fc in FIGS. 21A -21E, and measuring tumor volume changesin the mice.

FIG. 24E are flow cytometry dot plots showing the proportion of memoryprecursor effector cells (KLRG1⁻IL-7R⁺) and short-lived effector cells(KLRG1⁻IL-7R⁺) among CD8⁺ T cells in the spleen isolated fromtumor-bearing mice sacrificed 3 days after the third administration inFIGS. 21A-21E.

FIGS. 24F-24G are graphs showing the results of flow cytometry performedto analyze the proportion of memory precursor effector cells(KLRGLIL-7R+) (FIG. 24F) and short-lived effector cells (KLRG1⁺IL-7R⁻)(FIG. 24G) among CD8⁺ T cells in the spleen isolated from tumor-bearingmice sacrificed 3 days after the third administration in FIGS. 21A-21E.

FIG. 25A is a graph showing the results of flow cytometry analysisperformed to measure the proportion of CD8⁺ T cells (which showed highexpression of the transcription factor T-bet that inhibits memory celldifferentiation) in spleen cells isolated from mice sacrificed 3 daysafter the third administration in FIGS. 21A-21E.

FIG. 25B is a graph showing the results of flow cytometry analysisperformed to measure the proportion of CD8⁺ T cells (which showed highexpression of Eomes and low expression of T-bet) in spleen cellsisolated from mice sacrificed 3 days after the third administration inFIGs . 21A-21E.

FIG. 25C is a graph showing the results of flow cytometry analysisperformed to measure the expression level of phosphorylated STAT4 inCD8⁺ T cells isolated from tumor draining (inguinal) lymph nodes at 24hours after intraperitoneally administering bi-mIL-12-Fc andmono-mIL-12-Fc once at a concentration equimolar to 1 μg rmIL-12 whenthe tumor volume in Balb/c mice transplanted with CT26^(HER2/Neu) cancercells reached 300 mm³.

FIG. 25D is a graph showing the results of flow cytometry analysisperformed to measure the proportion of CD8⁺ T cells (which expressedT-bet that inhibits memory cell differentiation) in tumor draining(inguinal) lymph nodes at 72 hours after the single intraperitonealadministration in FIG. 25C.

FIG. 25E are histograms from flow cytometry analysis performed tomeasure the expression level of pSTAT4 when CD8⁺ T cells isolated fromthe spleen and inguinal lymph node of normal Balb/c mice were stimulatedwith the mono-mIL-12-Fc and bi-mIL-12-Fc that cross-reacted with anti-Fcantibody. FIG. 25F is a graph showing the results of flow cytometryanalysis performed to measure the expression level of pSTAT4 when CD8⁺ Tcells isolated from the spleen and inguinal lymph node of normal Balb/cmice were stimulated with the mono-mIL-12-Fc and bi-mIL-12-Fc thatcross-reacted with anti-Fc antibody.

FIG. 25G are flow cytometry dot plots showing the proportion ofT-bet-expressing CD8⁺ T cells when CD8⁺ T cells isolated from the spleenand groin lymph node of normal Balb/c mice were stimulated with themono-mIL-12-Fc and bi-mIL-12-Fc that cross-reacted with anti-Fcantibody. FIG. 25H is a graph showing the results of flow cytometryanalysis performed to measure the proportion of T-bet-expressing CD8⁺ Tcells when CD8⁺ T cells isolated from the spleen and groin lymph node ofnormal Balb/c mice were stimulated with the mono-mIL-12-Fc andbi-mIL-12-Fc that cross-reacted with anti-Fc antibody.

FIG. 26 is an overall schematic view showing a mechanism that inducesdifferentiation of memory precursor effector cells by mono-mIL-12-Fe anda mechanism that induces differentiation of short-lived effector cellsby bi-mIL-12-Fe.

BEST MODE FOR CARRYING OUT THE INVENTION

Unless defined otherwise, all the technical and scientific terms usedherein have the same meaning as those generally understood by one ofordinary skill in the art to which the invention pertains. Generally,the nomenclature used herein and the experiment methods, which will bedescribed below, are those well-known and commonly employed in the art.

In one aspect, the present invention relates to a heterodimeric Fc-fusedprotein comprising a first Fc region and a second Fc region of animmunoglobulin Fc pair and a physiologically active protein, wherein thephysiologically active protein is composed of two or more differentsubunits, wherein the two or more different subunits exhibitphysiological activity by forming a protein complex, wherein one or moresubunits of a physiologically active protein are linked to one or moreends of the N-terminus or C-terminus of the first Fc region and/or thesecond Fc region, wherein CH3 domains of the first Fc region and thesecond Fc region are mutated so as to promote heterodimer formation.

As used herein, the term “Fc region” or “heavy chain constant region”means a region comprising an immunoglobulin CH2 domain, a CH3 domain anda hinge domain. However, for IgE, the term means a region comprising aCH2 domain, a CH3 domain, a CH4 domain and a hinge domain.

As used herein, the expression “the first Fc region and the second Fcregion are mutated so as to promote heterodimer formation” means that anaturally occurring antibody has a homodimeric form in which two Fcregions have the same sequence, and a portion of these Fc regionsequences is mutated, so that heterodimer formation can be promotedthrough a specific non-covalent interaction between the first Fc regionand the second Fc region, or homodimer formation can be reduced, orpreferably can hardly occur.

Preferably, “the first Fc region and the second Fc region are mutated soas to promote heterodimer formation” may include “each of CH3 domainscontained in the first Fc region and second Fc region fromimmunoglobulin is mutated so as to promoter Fc heterodimer formation”.

In the present invention, “heterodimeric Fc or Fc heterodimer” comprisesthe first Fc region and the second Fc region, and the first Fc regionand the second Fc region mean heterodimers in which CH3 domains of thefirst Fc region and the second Fc region are mutated so as to promote Fcheterodimer formation.

In the present invention, each of the first Fc region and the second Fcregion may be derived from an Fc region selected from the groupconsisting of human IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD and IgE, andpreferably each of the first Fc region and the second Fc region isderived from IgG1, IgG2, IgG3 or IgG4.

In addition, the first Fc region and the second Fc region may be derivedfrom an isotype antibody.

In another aspect, the mutation of CH3 domain may include one or moremutations selected from the following group (wherein all mutationpositions in the present invention are numbered according to the EUindex): (1) substitution of the amino acid residue at position K370 inthe CH3 domain of the first Fc region; and substitution of the aminoacid residue at position(s) E357 and/or S364 in the CH3 domain of thesecond Fc region; and/or (2) substitution of the amino acid residue atposition K409 in the CH3 domain of the first Fc region; and substitutionof the amino acid residue at position(s) F405 and/or D399 in the CH3domain of the second Fc region.

Preferably, the substitution of amino acid residue at position K370 inthe CH3 domain of the first Fc region may be K370E, K370R, K370M, K370Dor K370H, substitution of the amino acid residue at position E357 in theCH3 domain of the second Fc region may be E357N, E357D, E357A, E357I,E357G or E357M, and substitution of the amino acid residue at positionS364 in the CH3 domain of the second Fc region may be S364T or S364W.

In addition, substitution of the amino acid residue at position K409 inthe CH3 domain of the first Fc region may be K409W, substitution of theamino acid residue at position F405 in the CH3 domain of the second Fcregion may be F405T, and substitution of the amino acid residue atposition D399 in the CH3 domain of the second Fc region may be D399V.

The amino acid residue mutation such as K370E means that K at position370 is mutated to E, and the mutation of all amino acid residues in thepresent invention is used as the same meaning as described above.

Most preferably, the mutation of the CH3 domain of the first Fc regionor the second Fc region may include one or more mutations selected fromthe following group (wherein mutation positions are numbered accordingto the EU index.): (1) a substitution K370E, K370R, K370M, K370D orK370H of the amino acid residue at position K370 in the CH3 domain ofthe first Fc region; (2) a substitution E357N, E357D, E357A, E357I,E357G or E357M of the amino acid residue at position E357 in the CH3domain of the second Fc region, and substitution S364T or S364W of theamino acid residue at position S364 in the CH3 domain of the second Fcregion; (3) a substitution K409W of the amino acid residue at positionK409 in the CH3 domain of the first Fc region; and (4) a substitutionF405T of the amino acid residue at position F405 in the CH3 domain ofthe second Fc region, and substitution D399V of the amino acid residueat position D399 in the CH3 domain of the second Fc region.

The CH3 domains in the first Fc region and the second Fc region mayfurther include the following residue: (i) cysteine (C) substituted atposition Y349 in the CH3 domain of the first Fc region; and (ii)cysteine (C) substituted at position S354 in the CH3 domain of thesecond Fc region.

In still another aspect, mutation of the CH3 domain may include one ormore mutations selected from the following group: (1) a substitution ofthe amino acid residue at position K360 in the CH3 domain of the firstFc region; and substitution of the amino acid residue at position E347in the CH3 domain of the second Fc region; and/or (2) a substitution ofthe amino acid residue at position K409 in the CH3 domain of the firstFc region; and substitution of the amino acid residue at position(s)F405 and D399 in the CH3 domain of the second Fc region.

Preferably, the substitution of the amino acid residue at position K360in the CH3 domain of the first Fc region may be K360E, and substitutionof the amino acid residue at position E347 in the CH3 domain of thesecond Fc region may be E347R.

Substitution of the amino acid residue at position K409 in the CH3domain of the first Fc region may be K409W, substitution of the aminoacid residue at position F405 in the CH3 domain of the second Fc regionmay be F405T, and substitution of the amino acid residue at positionD399 in the CH3 domain of the second Fc region may be D399V.

Most preferably, the mutation of the CH3 domain of the first Fc regionor the second Fc region may include one or more mutations selected fromthe following group (wherein mutation positions are numbered accordingto the EU index): (1) a substitution K360E of the amino acid residue atposition K360 in the CH3 domain of the first Fc region; (2) asubstitution E347R of the amino acid residue at position E347 in the CH3domain of the second Fc region; (3) a substitution K409W of the aminoacid residue at position K409 in the CH3 domain of the first Fc region;and (4) a substitution F405T of the amino acid residue at position F405in the CH3 domain of the second Fc region, and substitution D399V of theamino acid residue at position D399 in the CH3 domain of the second Fcregion.

The CH3 domains in the first Fc region and the second Fc region mayfurther include the following residue: (i) cysteine (C) substituted atposition Y349 in the CH3 domain of the first Fc region; and (ii)cysteine (C) substituted at position S354 in the CH3 domain of thesecond Fc region.

Preferably, each of the CH3 domains contained in the first Fc region andthe second Fc region from immunoglobulin according to the presentinvention may have an amino acid sequence selected from the groupconsisting of the amino acid sequences represented by the following SEQID NOS: (1) SEQ ID NO: 1 and SEQ ID NO: 2; (2) SEQ ID NO: 3 and SEQ IDNO: 4; (3) SEQ ID NO: 5 and SEQ ID NO: 6; (4) SEQ ID NO: 8 and SEQ IDNO: 9; (5) SEQ ID NO: 11 and SEQ ID NO: 12; and (6) SEQ ID NO: 14 andSEQ ID NO: 15.

In particular, the first Fc region and second Fc region fromimmunoglobulin according to the present invention preferably have thesequences of IgG4 CH3 domains shown in Table 1 below.

TABLE 1 con- CH3 sequence of  CH3 sequence of  figur-first Fc region (EU second Fc region (EU  ation number 341 to 447)number 341 to 447) γ4-  GQPREPQVYTLPPSQEEMT GQPREPRVYTLPPSQEEMTKNQ EWRVTENQVSLTCLVKGFYPSDIA VSLTCLVKGFYPSDIAVEWESN VEWESNGQPENNYKTTPPVGQPENNYKTTPPVLVSDGSFTL LDSDGSFFLYSWLTVDKSR YSRLTVDKSRWQEGNVFSCSVMWQEGNVFSCSVMHEALHNH HEALHNHYTQKSLSLSLGK YTQKSLSLSLGK (SEQ ID NO: 2)(SEQ ID NO: 1) γ4-  GQPREPQVCTLPPSQEEMT GQPREPRVYTLPPCQEEMTKNQEWRVT_(s-s) ENQVSLTCLVKGFYPSDIA VSLTCLVKGFYPSDIAVEWESNVEWESNGQPENNYKTTPPV GQPENNYKTTPPVLVSDGSFTL LDSDGSFFLYSWLTVDKSRYSRLTVDKSRWQEGNVFSCSVM WQEGNVFSCSVMHEALHNH HEALHNHYTQKSLSLSLGKYTQKSLSLSLGK (SEQ ID NO: 4) (SEQ ID NO: 3) γ4-  GQPREPQVYTLPPSQEEMTGQPREPQVYTLPPSQENMTKNQ A107 KNQVSLTCLVEGFYPSDIA VSLTCLVKGFYPSDIAVEWESNVEWESNGQPENNYKTTPPV GQPENNYKTTPPVLVSDGSFTL LDSDGSFFLYSWLTVDKSRYSRLTVDKSRWQEGNVFSCSVM WQEGNVFSCSVMHEALHNH HEALHNHYTQKSLSLSLGKYTQKSLSLSLGK (SEQ ID NO: 6) (SEQ ID NO: 5)

In the heterodimeric Fc-fused protein according to the presentinvention, a subunit of the physiologically active protein may be linkedonly to any one end of the N-terminus or C-terminus of the first Fcregion or the second Fc region, and one or more different subunits of asingle physiologically active protein may be linked to each of theN-terminus and C-terminus of each of the first Fc region and the secondFc region (see FIGS. 2B and 2C).

“A subunit of the physiologically active protein is linked only to anyone end of the N-terminus or C-terminus of the first Fc region or thesecond Fc region” means that one of the subunits of the physiologicallyactive protein is linked only to any one of four ends of the N-terminusor C-terminus of the first Fc region or the second Fc region, and theremaining subunit(s) of the physiologically active protein is(are)linked by a linker to the subunit of physiologically active protein,which is linked to any one end of the N-terminus or C-terminus of thefirst Fc region or the second Fc region. The linker is preferably anamino acid linker, but is not limited thereto.

In addition, “one or more different subunits of a single physiologicallyactive protein are linked to each of the N-terminus and C-terminus ofeach of the first Fc region and the second Fc region” means that one ormore different subunits of a single physiologically active protein arelinked to the N-terminus of each of the first Fc region and the secondFc region, one or more different subunits of a single physiologicallyactive protein are linked to the C-terminus of each of the first Fcregion and the second Fc region, or one or more different subunits of asingle physiologically active protein are respectively linked to theN-terminus and C-terminus of each of the first Fc region and the secondFc region.

In the heterodimeric Fc-fused protein according to the presentinvention, the subunit of the physiologically active protein may belinked to the N-terminus and/or C-terminus of the first Fc region and/orthe second Fc region by genetic fusion.

In still another aspect, the subunit of the physiologically activeprotein may be linked to the first Fc region and the second Fc regionthrough a linker. The linker is preferably an amino acid linker, but isnot limited thereto.

In yet another aspect, in the heterodimeric Fc-fused protein accordingto the present invention, the physiologically active protein ischaracterized in that it is composed of two or more different subunits,wherein the two or more different subunits exhibit physiologicalactivity by forming a protein complex.

“The physiologically active protein is composed of two or more differentsubunits, wherein the two or more different subunits exhibitphysiological activity by forming a protein complex” means that thephysiologically active protein exhibits desired physiological activitywhen two or more subunits form a protein complex.

The physiologically active protein is selected from the group consistingof interleukin-12 (IL-12), interleukin-23 (IL-23), interleukin-27(IL-27), interleukin-35 (IL-35), and follicle stimulating hormone (FSH),but is not limited thereto. Besides, it will be obvious to those skilledin the art that any physiologically active protein suitable for thepurpose of the present invention may be used in the present invention.

Most preferably, the physiologically active protein according to thepresent invention is IL-12. A protein which is composed of two or moretwo different subunits, wherein the two or more different subunitsexhibit physiological activity by forming a protein complex according tothe present invention will now be described in detail by way of exampleof IL-12 which is a preferred physiologically active protein.

IL-12 is composed of two subunits, p35 (IL-12A) and p40 (IL-12B), andthe physiologically active form of IL-12 is p70 which is a heterodimerof p35 and p40. IL-12 should be present in the form of p70 which is theheterodimer of p35 and p40 in order for IL-12 to exhibit the activitythereof in nature systems.

In the present invention, in order to mimic the form of naturallyoccurring IL-12 to the greatest possible extent, the form of aheterodimeric Fc-fused protein according to the present invention wasembodied.

Specifically, as described above, in the heterodimeric Fc-fused proteincomprising a first Fc region and a second Fc region according to thepresent invention, wherein one or more subunits of a physiologicallyactive protein are linked to one or more ends of the N-terminus orC-terminus of the first Fc region and the second Fc region, (i) one ormore subunits constituting a physiologically active protein may belinked only to any one end of the N-terminus or C-terminus of the firstFc region or the second Fc region, and the remaining subunit(s) of thephysiologically active protein may be linked by a linker, or (ii) one ormore different subunits of a single physiologically active protein maybe respectively linked to the N-terminus and/or C-terminus of each ofthe first Fc region and the second Fc region”.

In the above case, an example of IL-12 will be described hereinafter.

In the case of (i), the p35 or p40 subunit of IL-12 may be linked onlyto any one end of the N-terminus or C-terminus of the first Fc region orthe second Fc region, and the remaining subunit may be linked by alinker to the p35 or p40 subunit linked to any one end of the N-terminusor C-terminus of the first Fc region or the second Fc region to form theheterodimeric Fc-fused protein (see FIGS. 2B, and 2C).

In the case of (ii), any one selected from the p35 and p40 subunits ofIL-12 may be linked only to the N-terminus or C-terminus of the first Fcregion, and the other subunit may be linked only to the N-terminus orC-terminus of the second Fc region to form the heterodimeric Fc-fusedprotein (see FIGS. 2B, and 2C).

It was found that this form showed in vitro physiological activitysimilar to that of a conventional recombinant IL-12 protein whilemaintaining the naturally occurring original heterodimeric form (seeFIGS. 2B, 2C, and 10C).

Accordingly, a preferable immunoglobulin heterodimeric Fc-fused proteinaccording to the present invention is characterized in that thephysiologically active protein is IL-12, and in that the p35 or p40subunit of IL-12 is linked only to any one end of the N-terminus orC-terminus of the first Fc region or the second Fc region, and theremaining subunit is linked by a linker to the subunit linked to any oneend of the N-terminus or C-terminus of the first Fc region or the secondFc region, or in that the p35 and p40 subunits of IL-12 are linked toeach of the N-terminus and C-terminus of each of the first Fc region andthe second Fc region.

In another aspect, in the heterodimeric Fc-fused protein according tothe present invention, the hinge domain included in the N-terminus ofeach of the first Fc region and the second Fc region may becharacterized in that the cysteine residues contained in the hingedomain is mutated.

Preferably, mutation of the cysteine residues in the hinge domain may becharacterized in that cysteine residues in an upper hinge region, otherthan cysteine residues in a core hinge domain for heterodimer formation,are all substituted with serine residues, but the scope of the presentinvention is not limited thereto.

In addition, on the present invention, the first Fc region and thesecond Fc region may be included in a whole antibody form consisting ofhuman IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD and IgE.

In the present invention, the term “whole antibody form” means an intactantibody further comprising a CH1 domain, a VH domain, a CL domain and aVL domain, in addition to the CH2 domain, CH3 domain and hinge domain(also comprising CH4 domain for IgE) in the Fc region for IgG, IgA andIgD.

In still another aspect, the present invention relates to apharmaceutical composition comprising the heterodimeric Fc-fused proteinaccording to the present invention. The use of the pharmaceuticalcomposition according to the present invention may depend on the use ofa physiologically active protein contained in the heterodimeric Fc-fusedprotein.

Preferably, the physiologically active protein contained in theheterodimeric Fc-fused protein according to the present invention may beIL-12 or one or more subunits thereof. Therefore, the present inventionprovides a pharmaceutical composition for treating cancer, whichcomprises a heterodimeric Fc-fused protein comprising IL-12 as aphysiologically active protein.

A cancer that can be treated with the pharmaceutical composition fortreating cancer, which comprises a heterodimeric Fc-fused proteincomprising IL-12 or one or more subunits as the physiologically activeprotein may be selected from the group consisting of colorectal cancer,melanoma, breast cancer, pancreatic cancer, kidney cancer, prostatecancer, ovarian cancer, small intestine cancer, esophageal cancer,cervical cancer, lung cancer, lymphoma, and blood cancer, but notlimited thereto.

A pharmaceutical composition according to the present invention mayfurther comprise a pharmaceutically acceptable carrier. The term“pharmaceutically acceptable carrier” refers to a substance which can beadded to the active ingredient to help formulate or stabilize thepreparation and causes no significant adverse toxicological effects tothe patient.

As used herein, the term “pharmaceutically acceptable carrier” refers toa carrier or diluent that does not impair the biological activity andcharacteristics of a heterodimeric Fc-fused protein according to thepresent invention without irritating a patient. As a pharmaceuticallyacceptable carrier in a composition that is formulated as a liquidsolution, a sterile and biocompatible carrier is used. Thepharmaceutically acceptable carrier may be physiological saline, sterilewater, Ringer's solution, buffered saline, albumin injection solution,dextrose solution, maltodextrin solution, glycerol, ethanol, or amixture of two or more thereof. In addition, the composition of thepresent invention may, if necessary, comprise other conventionaladditives, including antioxidants, buffers, and bacteriostatic agents.Further, the composition of the present invention may be formulated asinjectable forms such as aqueous solutions, suspensions or emulsionswith the aid of diluents, dispersants, surfactants, binders andlubricants. In addition, the composition according to the presentinvention may be formulated in the form of pills, capsules, granules, ortablets. Other carriers are described in a literature [Remington'sPharmaceutical Sciences (E. W. Martin)].

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. The use of such media andagents for pharmaceutically active substances is known in the art. Thecomposition is preferably formulated for parenteral injection. Thecomposition can be formulated as a solid, a solution, a microemulsion, aliposome, or other ordered structures suitable to high drugconcentration. The carrier may be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol and liquid polyethylene glycol), and suitable mixturesthereof. In some cases, the composition may contain an isotonic agent,for example, sugar, polyalcohol, for example, sorbitol or sodiumchloride. Sterile injectable solutions can be prepared by theheterodimeric Fc-fused protein in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by sterile microfiltration. Generally, dispersionsare prepared by incorporating an active compound into a sterile vehicle,which contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying, which yielda powder of the active ingredient and any additional desired ingredientfrom a previously sterile-filtered solution thereof.

In addition, the pharmaceutical composition according to the presentinvention may be orally or parenterally administered to sufferingpatients at a dosage and frequency that may vary with the severity ofthe suffering patients. The compositions may be administered to patientsin need as a bolus or by continuous infusion. In another example, thepharmaceutical composition according to the present invention may beadministered rectally, intravenously, subcutaneously, intrauterinely, orintracerebrovascularly, but is not limited thereto.

In addition, a pharmaceutical composition for cancer treatment,comprising an immunoglobulin heterodimeric Fc-fused protein includingIL-12 can be used for combination therapy with other anticancer drugs.Other anticancer drugs are preferably cytotoxic T cells and/or naturalkiller (NK) cells, but not limited thereto, and all the anticancer drugsthat can be used in the art to which the present invention pertains canbe used for the combination therapy.

In particular, when a pharmaceutical composition for cancer treatment,comprising an immunoglobulin heterodimeric Fc-fused protein includingIL-12, is used for combination therapy with cytotoxic T cells and/ornatural killer (NK) cells, it may induce: (1) an increase in cytokinesecretion by stimulation of the T cells or natural killer (NK) cells;(2) an increase in antibody-dependent cell-mediated cytotoxicity (ADCC)or cytotoxic T lymphocyte (CTL) response; (3) an increase in the numberof cytotoxic T lymphocytes (CTLs) and/or natural killer cells; (3) anincrease in lymphocyte introduction around a tumor; or (4) an increasein the IL-12R betal and IL-12R beta2 signaling of lymphocytes in vivo.

In yet another aspect, the present invention relates to a method fortreating or preventing diseases, comprising administering, to a patientin need of treatment, a pharmaceutical composition comprising theheterodimeric Fc-fused protein according to the present invention.

Similar to the case of the composition, a disease that can be treated orprevented depends on the use of a physiologically active proteincontained in the heterodimeric Fc-fused protein. Preferably, when one ormore subunits of a physiologically active protein contained in theheterodimeric Fc-fused protein according to the present invention areone or more subunits of IL-12, the present invention provides a cancertreatment or prevention method for a patient suffering from a cancer,particularly a cancer selected from the group consisting of colorectalcancer, melanoma, breast cancer, pancreatic cancer, kidney cancer,prostate cancer, ovarian cancer, small intestine cancer, esophagealcancer, cervical cancer, lung cancer, lymphoma, and blood cancer.

EXAMPLES

Hereinafter, the present invention will be described in further detailwith reference to examples. It will be obvious to a person havingordinary skill in the art that these examples are for illustrativepurposes only and are not to be construed to limit the scope of thepresent invention.

Example 1 Design of Antibody Fc CH3 Domain Variants for HeterodimerFormation for Each Human Immunoglobulin Isotype (Sequencing)

In order to make heterodimeric Fc fragments for each humanimmunoglobulin isotype by introducing CH3 domain mutations that flavorheterodimer formation, the amino acid sequence similarity between CH3domains playing a major role in interactions for heterodimer formationwas first analyzed as described below. In this regard, the heterodimericFc variant (A107) was generated by introducing asymmetric mutations intothe CH3 homodimeric interface to generate heterodimeric CH3A:CH3B pair(in the present invention, CH3A and CH3B mean the CH3 domain of thefirst Fc region and the CH3 region of the second Fc region,respectively) by a strategy as published in previous literature orpatent documents (Choi et al. 2016; Korean Patent Application No.2015-0142181), such that the heterodimerization of CH3A:CH3B drive theFc variant to form the heterodimer in high yield. FIG. 3 aligns andcompares the sequences of CH3 domains for each human antibodyimmunoglobulin G (IgG) isotype. Each amino acid sequence was obtainedfrom the International ImMunoGeneTics information system (IMGT; URL:http://www.imgt.org/). In particular, among various allotypes, thesequence of G3m(s,t) whose serum half-life was reported to be maintainedat levels similar to those of other IgG isotypes was used for IgG3(Stapleton N M et al., 2011).

The results of sequencing indicated that IgG4 has a sequence conservedin all isotypes, except that the amino acid at position 409 amongpositions into which the A107 mutation is introduced is arginine, unlikethose in IgG1, IgG2 and IgG3. Accordingly, positions having the sameamino acid sequence number were selected as positions for introducingthe A107 mutation pair into isotypes other than IgG1. All amino acidpositions in the present invention are numbered according to the EUindex.

Example 2 Design of Immunoglobulin Fc CH3 Domain Variants forHeterodimer Formation for Each Human Immunoglobulin Isotype (StructuralModeling)

Before CH3 domain variants for each isotype were actually constructed,whether the A107 mutation pair could be stably introduced into thepositions selected in Example 1 so as to form heterodimers was predictedthrough structural modeling using the variant sequences introduced witheach mutation as shown in FIG. 3. Structural modeling was predictedthrough an online modeling server (URL: https://swissmodel.expasy.org/;Biasini M et al., 2014) using an already known immunoglobulin Fcheterodimer variant structure (PDB ID: 4X98) as a template. Each of theobtained structures was overlapped using the Pymol software, which couldvisualize protein structures, in order to observe the structural changeof the CH3 domain and the position of the A107 mutation afterintroduction of the mutation. On the overlapping structure, it was seenthat, even when the A107 mutation was introduced into each isotype, thestructures were maintained without great changes, compared to themodeled structure of conventional A107 variants constructed based onIgG1 isotypes and forming CH3A:CH3B Fc heterodimers. In particular, itwas shown that the directions of the introduced A107 mutation amino acidresidues were almost consistent and that the distances for interactionbetween the mutated amino acids were also maintained at similar levels(see FIG. 4).

Example 3 Construction of A107 Heterodimeric Fc Isotype Variants forEach Human Immunoglobulin Isotype

The A107 heterodimeric Fc isotype variants designed through thesequencing in Example 1 and the structural modeling in Example 2 werecloned in-frame into the animal cell expression vectorpcDNA3.1(+)(Invitrogen, USA) to have signal sequence-hinge-CH2-CH3 usingNotI/HindIII restriction enzymes and synthetic oligonucleotides(Macrogen, Korea) by a site-directed mutagenesis method which isperformed by those skilled in the art (see FIG. 5).

In the hinge domain used, the cysteine residues in the upper hingeregion, other than the cysteine residues in the core hinge region forheterodimer formation, were substituted with serine residues in order toprevent disulfide bonds from being produced during protein fusion. Inparticular, for IgG3, it was found in the literature that the highantibody effector functions (ADCC and CDC) of IgG3 are maintained evenby only the C-terminal 15 amino acids of the core hinge domain among the47 amino acids of the hinge domain of the G3m(s,t) allotype (Dall'AcquaW F et al., 2006). Accordingly, only the C-terminal 15 amino acids ofthe sequence shown in FIG. 5 were used.

Table 2 below shows the amino acid sequence information of the CH3regions in the wild-type and A107 heterodimeric Fc variant pairs of thepresent invention.

TABLE 2 CH3A chain CH3B chain con- (CH3 sequence of (CH3 sequence offigur- first Fc region) (EU second Fc region) (EU ationnumber 341 to 447) number 341 to 447) IgG1 GQPREPQVYTLPPSRDELTKNSame as SEQ ID NO: 7 Wild QVSLTCLVKGFYPSDIAVEWE typeSNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSP GK(SEQ ID NO: 7) γ1-A107 GQPREPQVYTLPPSRDELTKN GQPREPQVYTLPPSRDNLTKNQVSLTCLVEGFYPSDIAVEWE QVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSSNGQPENNYKTTPPVLVSDGS FFLYSWLTVDKSRWQQGNVFS FTLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP CSVMHEALHNHYTQKSLSLSP GK GK (SEQ ID NO: 8)(SEQ ID NO: 9) IgG2 GQPREPQVYTLPPSREEMTKN Same as SEQ ID NO: 10 WildQVSLTCLVKGFYPSDIAVEWE type SNGQPENNYKTTPPMLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO: 10) γ2-A107 GQPREPQVYTLPPSREEMTKNGQPREPQVYTLPPSRENMTKN QVSLTCLVEGFYPSDIAVEWE QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS SNGQPENNYKTTPPMLVSDGS FFLYSWLTVDKSRWQQGNVFSFTLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSP CSVMHEALHNHYTQKSLSLSP GK GK(SEQ ID NO: 11) (SEQ ID NO: 12) IgG3 GQPREPQVYTLPPSREEMTKNSame as SEQ ID NO: 13 Wild QVSLTCLVKGFYPSDIAMEWE typeSSGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNIFS CSVMHEALHNHYTQKSLSLSP GK(SEQ ID NO: 13) γ3-A107 GQPREPQVYTLPPSREEMTKN GQPREPQVYTLPPSRENMTKNQVSLTCLVEGFYPSDIAMEWE QVSLTCLVKGFYPSDIAMEWE SSGQPENNYKTTPPVLDSDGSSSGQPENNYKTTPPVLVSDGS FFLYSWLTVDKSRWQQGNIFS FTLYSKLTVDKSRWQQGNIFSCSVMHEALHNHYTQKSLSLSP CSVMHEALHNHYTQKSLSLSP GK GK (SEQ ID NO: 14)(SEQ ID NO: 15) IgG4 GQPREPQVYTLPPSQEEMTKN Same as SEQ ID NO: 16 WildQVSLTCLVKGFYPSDIAVEWE type SNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK (SEQ ID NO: 16) γ4-A107 GQPREPQVYTLPPSQEEMTKNGQPREPQVYTLPPSQENMTKN QVSLTCLVEGFYPSDIAVEWE QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS SNGQPENNYKTTPPVLVSDGS FFLYSWLTVDKSRWQEGNVFSFTLYSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSL CSVMHEALHNHYTQKSLSLSL GK GK(SEQ ID NO: 5) (SEQ ID NO: 6)

Example 4 Evaluation of the Heterodimerization Ability of A107Heterodimeric Fc Variants for Each Human Immunoglobulin Isotype

In order to examine whether the A107 heterodimeric Fc isotype variantsconstructed in Example 3 actually have heterodimerization abilitysimilar to those of wild-type A107 variants, a scFv-Fc_(CH3A)/Fc_(CH3B)expression system which is mainly used to evaluate heterodimerizationability of Fc variants in the same kind of studies was used (Choi etal., 2013). FIG. 6 is a schematic view showing thescFv-Fc_(CH3A)/Fc_(CH3B) expression system. Since antibodies purified inthe scFv-Fc_(CH3A)/Fc_(CH3B) expression system show molecular weightsdifferent between an scFv-Fc_(CH3A) homodimer (103 kDa), anscFv-Fc_(CH3A)/Fc_(CH3B) heterodimer (78 kDa) and an Fc_(CH3B) homodimer(53 kDa), the degree of formation of the heterodimer can be compared onSDS-PAGE.

As the Fc_(CH3B) vector, the vector constructed in Example 3 was used.Additionally, a vector was cloned by introducing scFv only into theN-terminus of Fc_(CH3A), that is, providing a format ofpcDNA3.1(+)-scFv-hinge-CH2-CH3A (scFv-Fc_(CH3A)). FIG. 7 is a schematicview of the animal cell expression vectorpcDNA3.1(+)-scFv-hinge-CH2-CH3A (scFv-Fc_(CH3A)) used in thescFv-Fc_(CH3A)/Fc_(CH3B) expression system. The scFv antibody used is anantibody obtained by linking the VH and VL regions of hAY4a which is anaffinity-enhanced version of the humanized antibody hAY4 that bindsspecifically to DR4 (Lee, Park et al. 2010). Cloning was performed usingNotl restriction enzyme and the BsiWI restriction enzyme locatedimmediately before the hinge domain. As a control for the variant,wild-type Fc was constructed in the same format (scFv-Fc/Fc).

Example 5 Expression and Purification of Antibodies Comprising A107Heterodimeric Fc Variants for Each Human Immunoglobulin Isotype

Co-expression of the constructed scFv-Fc_(CH3A) and Fc_(CH3B) wasperformed by transiently transfecting a mixture of expression vectors(1:1 ratio) and polyethylenimine (PEI) (Polyscience) into HEK293-F cells(Invitrogen) and culturing the cells in a shake flask containingserum-free FreeStyle 293 expression medium. The detailed method is asfollows.

For 200 mL transfection in a shake flask (Corning), HEK293-F cells wereseeded into 100 ml of medium at a density of 2.0×10⁶ cells/ml, andcultured at 150 rpm under 8% CO₂. To produce each humanized antibody,heavy chain and light-chain plasmids for each antibody were diluted in10 ml of FreeStyle 293 expression medium (Invitrogen) in a total amountof 250m (2.5 μg/ml) (125 μg for the light chain and 125 μg for the heavychain), and the medium was mixed with 10 ml of medium (7.5m/ml)containing 750 m of PEI diluted therein. The medium mixture wasincubated at room temperature for 10 minutes. Next, the incubated mediummixture was added to 100 ml of the seeded cells and incubated for 4hours at 150 rpm under 8% CO₂, after which the remaining 100 ml ofFreeStyle 293 expression medium was added thereto, followed byincubation for 5 to 7 days. During this incubation, the protein producedby the cells, that is, an antibody comprising the Fc variant, wassecreted out of the cells and accumulated in the medium. For thisreason, the protein was purified using the protein A Sepharose column(GE Healthcare) from the cell culture supernatant collected by 20minutes of centrifugation at 2500 rpm after cell culturing. In thiscase, the purification process was performed with reference to thestandard protocol provided by the protein A column company. The purifiedprotein was quantified by measuring the absorbance at a wavelength of562 nm using the solution in the BCA protein assay kit (Thermo) anddetermining the amount thereof according to the prepared standard curve.

Example 6 Evaluation of the Heterodimerization Ability of A107Heterodimeric Fc Variants for Each Human Immunoglobulin Isotype

5 μg of the antibody, purified in Example 5 and comprising the A107heterodimeric Fc variant for each isotype, was analyzed on 12% SDS-PAGEunder non-reducing conditions (FIG.

8). A homodimer of the CH3A variant was observed at 103 kD; a homodimerof the CH3B variant was observed at 53kD; a monomer of the CH3B variantwas observed at 25 kD; and a heterodimer of the CH3A variant and theCH3B variant was observed at 78 kD. To more accurately examine thedegree of homodimerization, Western blotting was also performed. Westernblotting was performed by isolating 0.1 μg of protein, which was smallerthan that in 12% SDS-PAGE analysis, under non-reducing conditions, andthen treating the protein with anti-human IgG-AP conjugated antibody(Sigma) according to a conventional method known in the art (FIG. 9).

As can be seen in FIGS. 8 and 9, for the IgG1 heterodimers introducedwith the wild-type CH3 domain which is a control, a homodimer of each ofCH3A and CH3B and a CH3A:CH3B heterodimer were all observed on SDS-PAGE,whereas the A107 heterodimeric Fc variants for each human immunoglobulinisotype, obtained by introducing the A107 heterodimerization mutationinto IgG2, IgG3 and IgG4, except for IgG1, all formed heterodimers inyields similar to or higher than those of previously reported IgG1-basedA107 variants. At this time, for the IgG4 variant, an Fc monomer (halfFc) comprising CH3A or CH3B was also observed, which is one of theproperties of naturally occurring IgG4 and results from the property offorming half Fc with respect to the hinge domain (particularly, serineat position 228 in the core hinge region) before the occurrence ofFab-arm exchange in blood (Liu H et al., 2012).

Example 7 Construction of Human/Mouse IL-12 Fusion Protein

The isotype variants in Examples 1 to 6 were found to retain theirheterodimerization ability at a level similar to that of the previouslyreported IgG1-based A107 heterodimeric Fc variant. Among these isotypevariants, the IgG4-based variant (γ4-A107) was used to construct along-lasting IL-12 fusion protein. Naturally occurring IL-12 is composedof two subunits, a p35 subunit (p35; IL-12A) and a p40 subunit (p40;IL-12B), and the two subunits interact to form a heterodimer havingactivity. Formation of this heterodimer is achieved because the twosubunits are more strongly and stably coupled by a single disulfide bondpresent between the two subunits. Accordingly, the two subunits (p35 andp40) of IL12 were genetically fused to the N-terminus of eachheterodimeric Fc chain in order to maintain the heterodimeric form ofthe naturally occurring cytokine.

As a heterodimeric Fc variant for construction of a fusion protein,γ4-A107 was used, which was based on IgG4 and would form a heterodimerby introduction of the A107 mutation. As previously reported, inconstruction of an immunocytokine which is a fusion of an antibody and acytokine, the antibody effector function (such as ADCC/CDC) of IgG1rather promotes in vivo clearance. For this reason, a fusion protein wasconstructed using an IgG4 isotype which hardly shows the ADCC/CDCfunction, compared to IgG1 (Gillies S D et al., 1999).

FIGS. 10A-10C show schematic views of an IL-12 recombinant protein (FIG.10A), a monovalent IL-12 fusion protein (mono-IL-12-Fc) obtained usingγ4-A107 (FIG. 10C), and a bivalent IL-12 fusion protein (bi-IL-12-Fc)obtained using wild-type Fc (FIG. 10B) in the present invention. Inparticular, FIG. 10C shows a fusion protein constructed by introducingthe CH3 variant pair in the present invention. The DNA sequence of eachof human IL-12 (hIL-12, Uniprot entry name P29460, P29459; SEQ ID NO:17-18) and mouse IL-12 (mIL-12, Uniprot entry name P43432, P43431; SEQID NO: 19-20), which encodes a mature form excluding a signal sequence,was amplified, and each amplification product was cloned in-frame intoan animal cell expression vector containing the γ4-A107 variant by useof NotI/B siWI restriction enzymes as shown in FIGS. 11A and 11B. Theresulting proteins were named mono-hIL-12-Fc and mono-mIL-12-Fc,respectively. In particular, a flexible peptide linker consisting of 15amino acids was added between the p35 subunit and the hinge domain sothat the human/mouse p35 subunit could sufficiently interact with thep40 subunit (flexible (G45)3 Linker). As comparative examples for theprotein shown in FIG. 10C, bi-hIL-12-Fc and bi-mIL-12-Fc wereconstructed by fusing each of human IL-12 (hIL-12) and mouse IL-12(mIL-12) to wild-type IgG4 Fc (wt IgG4). In order to fuse a single Fcwith IL-12 which have activity only in a heterodimeric form, the twosubunits of IL-12 were linked to each other by the 15-amino-acid peptidelinker, and then cloned in-frame into an animal cell expression vectorcontaining the γ4-A107 variant by use of NotI/BsiWI restriction enzymesas shown in FIG. 12. The comparative examples are fusion proteins usedin previous studies to make IL-12 fusion proteins (Lisan S. Peng et al.,1999).

Table 3 below shows the amino acid sequences for a mature form of thesubunits of the human and mouse IL-12 used for construction of thefusion proteins.

TABLE 3 con- figur- CH3A chain CH3B chain ation (p40 subunit)(p35 subunit) Mature IWELKKDVYVVELDWYPDA RNLPVATPDPGMFPCLHHSQN humanPGEMVVLTCDTPEEDGITW LLRAVSNMLQKARQTLEFYPC IL-12 TLDQSSEVLGSGKTLTIRVTSEEIDHVDITKDKTSTVEAC KEFGDAGQYTCHKGGEVLS LPLELTKNESCLNSRETSFITHSLLLLHKKEDGIWSTDIL NGSCLASRKTSFMMALCLSSI KDQKEPKNKTFLRCEAKNYYEDLKMYQVEFKTMNAKLLMD SGRFTCWWLTTISTDLTFS PKRQIFLDQNMLAVIDELMQAVKSSRGSSDPQGVTCGAAT LNFNSETVPQKSSLEEPDFYK LSAERVRGDNKEYEYSVECTKIKLCILLHAFRIRAVTIDR QEDSACPAAEESLPIEVMV VMSYLNAS DAVHKLKYENYTSSFFIRD(SEQ ID NO: 18) IIKPDPPKNLQLKPLKNSR QVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDR VFTDKTSATVICRKNASIS VRAQDRYYSSSWSEWASVP CS(SEQ ID NO: 17) Mature  MWELEKDVYVVEVDWTPDA RVIPVSGPARCLSQSRNLLKTTDmouse PGETVNLTCDTPEEDDITW DMVKTAREKLKHYSCTAEDIDHE IL-12TSDQRHGVIGSGKTLTITV DITRDQTSTLKTCLPLELHKNES KEFLDAGQYTCHKGGETLSCLATRETSSTTRGSCLPPQKTSL HSHLLLHKKENGIWSTEIL MMTLCLGSIYEDLKMYQTEFQAIKNFKNKTFLKCEAPNYSGR NAALQNHNHQQIILDKGMLVAID FTCSWLVQRNMDLKFNIKSELMQSLNHNGETLRQKPPVGEAD SSSPPDSRAVTCGMASLSA PYRVKMKLCILLHAFSTRVVTINEKVTLDQRDYEKYSVSCQE RVMGYLSSA DVTCPTAEETLPIELALEA (SEQ ID NO: 20)RQQNKYENYSTSFFIRDII KPDPPKNLQMKPLKNSQVE VSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETKEGC NQKGAFLVEKTSTEVQCKG GNVCVQAQDRYYNSSCSKW ACVPCRVRS(SEQ ID NO: 19)

Example 8 Expression/Purification of IL-12 Fusion Protein

The mono-IL-12-Fc fusion protein in FIG. 10C was expressed/purified fromthe human/mouse IL-12.p40-γ4-A107A and human/mouse IL-12.p35-γ4-A107Bexpression vectors (1:1 ratio) according to the method described inExample 5. The bi-IL-12-Fc fusion protein in FIG. 10B wasexpressed/purified through single transfection of the human/mousescIL-12-IgG4 Fc(wt) expression vector. All the fusion proteins wereexpressed/purified in an amount of 12 to 13 mg per 100 ml of the HEK293Fcell culture.

5 μg of each of the purified mono-IL-12-Fc and bi-IL-12-Fc fusionproteins was analyzed on 12% SDS-PAGE under non-reducing conditions(FIG. 13). A monomer of the IL-12.p40-CH3A variant was observed at 60kD; a homodimer of the IL-12.p40-CH3A variant was observed at 120 kD; amonomer of the IL-12.p35-CH3B variant was observed at 50 kD; a homodimerof the IL-12.p35-CH3B variant was observed at 100 kD; and a heterodimerof the IL-12.p40-CH3A variant and the IL-12.p35-CH3B variant wasobserved at 110 kD. However, for the proteins obtained by linking thehuman and mouse interleukin subunits, bands were observed at slightlydifferent sizes, and it was found in the literature that these bandsresult from different glycosylation patterns (Lo et al., 2007). Inaddition, in the same manner as described in Example 6 above, a monomerwas observed in all the IL-12 fusion proteins based on IgG4. Similar tothe previous report that the p35 subunit is not naturally expressed in amonomeric form without the aid of the p40 subunit, only a p40subunit-linked CH3A monomer was observed in the mono-IL-12-Fc fusionprotein obtained using the heterodimeric Fc variant (Gillies et al.,1998b).

FIG. 14 shows the results of analyzing the fusion proteins bysize-exclusion chromatography (SEC). An oligomer was partially observedfrom the Mono-hIL-12-Fc fusion protein.

Example 9 Evaluation of the Binding Affinity of Mono-hIL-12-Fc FusionProtein for IL-12 Receptor

The binding affinity of the mono-hIL-12-Fc, expressed and purified inExample 8, for the IL-12 receptor, was analyzed comparatively with thatof bi-hIL-12-Fc.

FIG. 15A and FIG. 15B show the results of FACS-Calibur (BD Biosciences)analysis performed to determine that the constructed mono-hIL-12-Fcwould show binding affinity for the IL-12 receptor, in comparison withbi-hIL-12-Fc.

Specifically, in order to isolate immune cells (PBMCs) from humanperipheral blood, 5 ml of Ficoll (GE Healthcare) was filled in a 15-mltest tube. Sampled blood was mixed with PBS (pH 7.4) at 1:1 and shaken,and then 10 ml of the blood was taken and centrifuged in theFicoll-containing test tube in a “no break” state at 750 g for 20minutes so as not to mix with Ficoll. Next, the buffy coat formed on theFicoll was recovered and washed twice with PBS (pH 7.4), and then PBMCs,including T cells, B cells, NK cells and monocytes, were obtained. Theisolated normal PBMCs did not express IL-12R in such large amounts thatthe binding of IL-12 could be observed (FIG. 15A). For this reason, thecells were stimulated by treatment with the mitogen PHA (Sigma-Aldrich)for 72 hours so that T cells and NK cells could be activated. It wasreported that when cells are treated with PHA, the IL-12 receptor isexpressed in T cells and NK cells while immune cells divide. PBMCs wereadded to 10% FBS-containing RPMI1640 medium at a density of 1×10⁶cells/ml, and the mitogen PHA was added thereto at a concentration of 10μg/ml, after which the cells were cultured in a 5% CO₂ incubator at 37°C. for 72 hours. Normal PBMCs and PHA-activated PBMCs were washed withcold PBS (pH 7.4), and 5×10⁵ cells per sample were prepared. Each of Fc(A107), bi-hIL-12-Fc and mono-hIL-12-Fc was added to each sample at aconcentration of 1 μM, incubated at 4° C. for 30 minutes, and thenwashed with cold PBS (pH 7.4). Each sample was incubated withFITC-conjugated human anti-IgG4 secondary antibody (Sigma-Aldrich) at 4°C. for 30 minutes, washed with PBS (pH 7.4), and then analyzed by flowcytometry (FACS Calibur, BD Bioscience). After analysis, a histogramgraph for each sample was obtained, and the binding affinity ofmono-hIL-12-Fc for the IL-12 receptor was evaluated.

The results of the analysis indicated that bi-hIL-12-Fc andmono-hIL-12-Fc did not bind to normal PBMCs expressing no IL-12 receptor(FIG. 15A) and did bind only to PHA-activated PBMCs expressing the IL-12receptor (FIG. 15B). Thus, it was found that the binding affinity ofmono-hIL-12-Fc for the IL-12 receptor was equal to that of bi-hIL-12-Fc.

Example 10 Evaluation of the Ability of Mono-hIL-12-Fc Fusion Protein toInduce PBMC Proliferation

Whether the IL-12 moiety in the IL-12 fusion protein would retainphysiological activity comparable to that actual recombinant IL-12(rIL-12) by its binding to the IL-12 receptor was examined usingrecombinant human IL-12 (rhIL-12, Thermo Fisher Scientific) as acontrol.

FIGS. 16(A)-16(D) show the results of a WST-1 cell proliferation assayperformed to examine the cell proliferation abilities of Fc (A107),rhIL-12, bi-hIL-12-Fc and mono-hIL-12-Fc in PHA-activated PBMCs.

Specifically, PBMCs (2×10⁴ cells, 50 μl) activated by PHA in the samemanner as described in Example 9 were added to a 96-well plate (SPL,Korea), followed by addition of 50 pl of each of 50-0.4 pM Fc (A107),rhIL-12, bi-hIL-12-Fc and mono-hIL-12-Fc diluted serially with 10%FBS-containing RPMI1640 medium. Next, the cells were cultured at 37° C.under 5% CO₂ for 72 hours. For a cell proliferation assay, 10 pl ofWST-1 (Water-soluble Tetrazolium salts, Sigma-aldrich) reagent was thenadded to each well and incubated at 37° C. for 4 hours, and theabsorbance at 570 nm was measured using a microplate reader (MolecularDevices).

As a result, it was shown that mono-hIL-12-Fc had a PBMC proliferationability similar to or higher than that of rhIL-12.

Example 11 Evaluation of the Ability of Mono-hIL-12-Fc Fusion Protein toInduce IFN-y Secretion from PBMCs

FIGS. 17(A)-17(D) show the results of an ELISA performed to measure theamount of IFN-γ secreted from PHA-activated PBMCs by Fc (A107), rhlL-12,bi-hIL-12-Fc and mono-hIL-12-Fc.

Specifically, in order to measure the concentration of IFN-γ in theculture supernatant cultured for 72 hours in Example 10, a 96-well plate(Thermo Fisher Scientific, Korea) for ELISA was coated with human IFN-γcapture antibody (Thermo Fisher Scientific) for 12 hours, washed withPBST, and then blocked with 1% BSA (PBS with 1% bovine serum albumin) atroom temperature for 1 hour. After washing with PBST (PBS with 0.1%Tween-20), the culture supernatant obtained in Example 2 was diluted5-fold with 1% BSA, and 100 μl of the dilution was added to each welland incubated at room temperature for 2 hour. After washing with PBST,each well was incubated with biotin-conjugated IFN-γ detection antibody(Thermo Fisher Scientific) at room temperature for 1 hour. After washingwith PB ST (PBS with 0.1% Tween-20), each well was incubated withavidin-conjugated horse radish peroxidase (HRP) (Thermo FisherScientific) at room temperature for 30 minutes, washed with PBST (PBSwith 0.1% Tween-20), and then treated with 3,3‘,5,5’-tetramethylbenzidine substrate (TMB, sigma-aldrich). Theabsorbance at 405 nm was measured using a microplate reader.

As a result, it was shown that the ability of mono-hIL-12-Fc to induceIFN-y secretion from PBMCs was similar to or higher than that ofrhIL-12.

Example 12 Evaluation of the Binding Affinity of Mono-mIL-12-Fc forIL-12 Receptor

The binding affinity of mono-mIL-12-Fc, expressed/purified in Example 8,for the IL-12 receptor, was analyzed comparatively with that ofbi-mIL-12-Fc.

FIGS. 18(A) and 18(B) show the results of flow cytometry performed todetermine that the constructed mono-mIL-12-Fc shows a binding affinityfor the IL-12 receptor, in comparison with bi-mIL-12-Fc.

Specifically, it was reported that mouse IL-12 binds not only to themouse IL-12 receptor, but also to the human IL-12 receptor. Thus,analysis was performed in the same manner as described in Example 9. Theresults of the analysis indicated that bi-mIL-12-Fc and mono-mIL-12-Fcdid not bind to normal PBMCs expressing no IL-12 receptor (FIG. 18A) anddid bind only to PHA-activated PBMCs expressing the IL-12 receptor (FIG.18B). Thus, it was shown that the binding affinity of mono-mIL-12-Fc forthe IL-12 receptor was the same as that of bi-mIL-12-Fc.

Example 13 Evaluation of the Ability of Mono-mIL-12-Fc to Induce PBMCProliferation

FIGS. 19(A) and 19(B) show the results of a WST-1 cell proliferationassay performed to examine effects of abilities of Fc (A107),recombinant mouse IL-12(rmIL-12), bi-mIL-12-Fc, and mono-mIL-12-Fc onthe cell proliferation of PHA-activated PBMCs.

Specifically, PBMCs (2×10⁴ cells, 50 μl) activated by PHA in the samemanner as described in Example 9 were added to a 96-well plate, followedby addition of 50 μl of each of 50-0.4 pM Fc (A107), rmIL-12,bi-mIL-12-Fc and mono-mIL-12-Fc diluted serially with 10% FBS-containingRPMI1640 medium. Next, the cells were cultured at 37° C. under 5% CO₂for 72 hours, and then a WST assay was performed in the same manner asdescribed in Example 10. As a result, it was shown that mono-mIL-12-Fchad the ability to induce PBMC proliferation, similar to rmIL-12.

Example 14 Evaluation of the Ability of Mono-mIL-12-Fc to Inhibit InVivo Tumor Growth

In Example 13, the ability of mono-mIL-12-Fc to induce the proliferationof PHA-activated PBMCs was evaluated. Whether the same effect ofmono-mIL-12-Fc would also appear in vivo was examined.

FIGS. 20A-20C show the results of measuring the tumor growth inhibitoryactivity of mono-mIL-12-Fc on 100 mm³ tumors in living mice.

Specifically, 4-week-old female Balb/c mice (NARA Biotech, Korea) wereshaved, and CT26^(HERS/Neu) colorectal cancer cells (1×10⁶ cells/mouse)diluted in 150 μL of PBS were transplanted subcutaneously into the mice.Mice having similar tumor volumes (average volume: 100 to 120 mm³) wererandomly grouped, and each of Fc (A107), rmlL-12 (Thermo FisherScientific), bi-mIL-12-Fc and mono-mIL-12-Fc was intraperitoneallyinjected a total of six times (twice a week) into each mouse at the dosecorresponding to an equivalent molar amount of 1 μg IL-12. The tumor wasmeasured twice a week, and the tumor volume (V) was calculated using thefollowing equation: V=length×width²/2.

As shown in FIG. 20A, in comparison with the control, administration of1 μg of rmIL-12 had no effect on the inhibition of tumor growth, but theequimolar concentrations of mono-mIL-12-Fc and bi-mIL-12-Fc inhibitedtumor growth. In addition, as shown in FIG. 20C, administration ofmono-mIL-12-Fc and bi-mIL-12-Fc showed little or no changes of mousebody weight compared to the control, indicating that mono-mIL-12-Fc andbi-mIL-12-Fc are not toxic.

FIGS. 21A-21N show the results of measuring the tumor growth inhibitoryactivity of various concentrations of mono-mIL-12-Fc on 300 mm³ tumorsin living mice.

Specifically, 4-week-old female Balb/c mice (NARA Biotech, Korea) wereshaved, and CT26^(HERS/Neu) colorectal cancer cells (1×10⁶ cells/mouse)diluted in 150 μL of PBS were transplanted subcutaneously into the mice.Mice having similar tumor volumes (average volume: 300 mm³) wererandomly grouped, and each of bi-mIL-12-Fc and mono-mIL-12-Fc wasintraperitoneally injected a total of 6 times (twice a week) into eachmouse at a concentration equimolar to 0.1-2 μg rmIL-12. The tumor wasmeasured twice a week, and the tumor volume (V) was calculated using thefollowing equation: V=length×width²/2.

As shown in FIGS. 21A-21N, at a dose corresponding to an equivalentmolar amount of 1 μg IL-12 (FIG. 21D) or less, mono-mIL-12-Fc showed ahigh effect of inhibiting the growth of large tumors, compared tobi-IL-12-Fc. At a concentration corresponding to an equivalent molaramount of 0.25 μg IL-12 (FIG. 21B), bi-mIL-12-Fc showed the effect ofinhibiting tumor growth, but did not remove the tumor. However, underthe identical dosing regimen, mono-mIL-12-Fc showed the effect ofremoving the tumor in 40% of the mice. In addition, at a concentrationcorresponding to an equivalent molar amount of 0.5 μg IL-12 (FIG. 21C)at which bi-mIL-12-Fc failed to remove the tumor, mono-mIL-12-Fc removedthe tumor in 73% of the mice even when it was administered only fivetimes.

Example 15 Evaluation of In Vivo Toxicity of Mono-mIL-12-Fc

FIG. 21O shows the results of measuring body weight changes to determinethe in vivo toxicity of mono-mIL-12-Fc administered at variousconcentrations.

Specifically, as shown in FIGS. 21A-21E, whether the body weight wouldbe reduced was observed by measuring the body weight of mice,administered with mono-mIL-12-Fc, twice a week. It was shown that thebody weight increased as the tumor volume increased in the controlgroup, but the mice administered with all concentrations of bi-mIL-12-Fc(FIG. 21P) and mono-mIL-12-Fc (FIG. 210) showed no decrease in the bodyweight compared to before administration. Thus, it was determined thatmono-mIL-12-Fc does not induce a reduction in body weight, and thus hasno significant in vivo toxicity.

FIG. 21Q shows the results of measuring alanine aminotransferase (ALT)that is a hepatotoxicity marker.

Specifically, blood was sampled from the facial veins of the mice ofFIGS. 21M-21N at 24 hours after the last administration. The blood wasallowed to stand at room temperature for 2 hours so as to induce bloodcoagulation, and then centrifuged at 8000 rpm for 10 minutes, and thesupernatant serum was collected. To measure the concentration of ALT inserum, blood was sampled from the mouse facial veins at 24 hours afterthe last administration of the IL-12-Fc fusion protein. The blood wasallowed to stand at room temperature for 2 hours so as to induce bloodcoagulation, and then centrifuged at 8000 rpm for 10 minutes, and thesupernatant serum was collected. To measure the concentration of ALT inthe serum, a substrate solution for ALT measurement (a mixture ofalanine and a-ketoglutarate) was taken in a 15-ml test tube andincubated in a constant-temperature water bath at 37° C. for 5 minutes.Serum isolated from the blood of tumor-transplanted mice administeredwith each of bi-mIL-12-Fc and mono-mIL-12-Fc was diluted 10-fold, and200 μl of the dilution was added to the substrate solution, shaken, andincubated in a constant-temperature water bath at 37° C. for 30 minutes.1 ml of a color development reagent (2,4-dinitrophenyl-1-hydrazone) wasadded to the test tube taken out from the constant-temperature waterbath, and the test tube was allowed to stand at room temperature for 20minutes. Next, 10 ml of 0.4 N sodium hydroxide solution was added to thetest tube and mixed, and then the test tube was allowed to stand at roomtemperature for 10 minutes. The absorbance at 505 nm was measured usinga photoelectric spectrophotometer (GeneQuant100, GE Healthcare). Using astandard curve prepared by adding a standard curve reagent instead ofserum, ALT was converted into units. It was shown that the serum fromthe blood sampled from mice administered with Bi-mIL-12-Fc ormono-mIL-12-Fc showed ALT activity similar to that of the serumseparated from the blood sample of the control or normal Balb/c mice.This suggests that when bi-mIL-12-Fc or mono-mIL-12-Fc is administeredto tumor-transplanted mice at a concentration equimolar to 0.5 μg or 1μgIL-12, it induces no hepatotoxicity.

Example 16 Evaluation of the Ability of mono-mIL-12-Fc to Induce ImmuneCell Proliferation In Vivo

As shown in Example 15, when bi-mIL-12-Fc and mono-mIL-12-Fc wereadministered at a concentration corresponding to an equivalent molaramount of 2 μg IL-12, bi-mIL-12-Fc and mono-mIL-12-Fc all removed thetumor, but when they were administered at a molar concentration lowerthan 1 μg IL-12, the tumor growth inhibitory effect of mono-mIL-12-Fcwas significantly higher than that of bi-mIL-12-Fc. In fact, analysiswas performed to determine whether the high tumor growth inhibitoryeffect of mono-mIL-12-Fc would be associated with an increase in thenumber of intrinsic effector cells such as NK cells, CD4⁺ T cells andCD8⁺ T cells, which have the IL-12 receptor.

FIGS. 22A-22C show the results of measuring increases in the number ofCD4⁺ T cells (FIG. 22A), CD8⁺ T cells (FIG. 22B), and NK cells (FIG.22C) in the spleen of mice sacrificed 3 days after the lastadministration in FIGS. 21A-21E.

Specifically, after treatment as shown in FIGS. 21A-21E, the mousespleen was dissected 34 days after tumor transplantation, crushed usinga wide mesh in a Petri dish, and then washed with 10 ml of 2%FBS-containing medium. Next, 1 ml of red blood cell lysis buffer wasadded thereto to lyse red blood cells, and the resulting cells werewashed with PBS to prepare a spleen cell suspension, and the number ofthe cells was countered with a hemocytometer. APC, FITC, PE orPE-cy5-conjugated anti-CD45, anti-CD3, anti-CD4, anti-CD8 and anti-CD49bantibodies were added to the spleen lymphocytes which were then stainedat 4° C. for 30 minutes, washed with cold PBS (pH 7.4), and thenanalyzed by flow cytometry (FACS Calibur, BD Bioscience) and Flow jo(Thermo Fisher Scientific). Each sample was analyzed by dot plots, andthe CD45⁺CD3⁺CD4⁺ cell population (FIG. 22A), the CD45⁺CD3⁺CD8⁺ cellpopulation (FIG. 22B) and the CD45⁺CD3⁻CD49b±cell population (FIG. 22C)were defined as CD4+T cells, CD8⁺ T cells and NK cells, respectively,and the proportions thereof relative to the total spleen cells werecalculated and multiplied by the cell number counted with ahemocytometer, and the number of CD4⁺ T cells, CD8⁺ T cells and NK cellswhich increased after administration of mono-mIL-12-Fc was analyzed.

As a result, it could be seen that, in comparison with the control,mono-mIL-12-Fc increased the number of CD4+ T cells (FIG. 22A) and CD8⁺T cells (FIG. 22B) in the tumor-transplanted mice in aconcentration-dependent manner. However, bi-mIL-12-Fc increased thenumber of CD8⁺ T cells only in the group administered with the same at aconcentration corresponding to an equivalent molar amount of 0.5 μgIL-12 (FIG. 22B), and it did not increase the number of CD4⁺ T cells(FIG. 22A) and CD8⁺ T cells (FIG. 22B) in the group administered withthe same at a concentration corresponding to an equivalent molar amountof 1 μg IL-12. Consistent with the previous study results (Cerwenka andLanier, 2016; Schreiber et al., 2011) that NK cells do not form memorycells in tumor-transplanted mice, it was observed that on 34 days aftertumor transplantation, the number of NK cells in the groups administeredwith mono-mIL-12-Fc and bi-mIL-12-Fc was similar to that in the controlgroup. As a result, it was shown that mono-mIL-12-Fc caused greaterexpansion of CD4+T cells and CD8⁺ T cells, accounting for the strongertumor growth inhibition, compared to bi-mIL12-Fc.

Based on the report (Schreiber et al., 2011) that an increase in thenumber of adaptive immune dells (CD4+T cells and CD8⁺ T cells) thatinfiltrated tumors is important in inhibiting tumor growth, whethermono-mIL-12-Fc would increase the number of adaptive immune cells thatinfiltrated tumors was analyzed. When mono-mIL-12-Fc was administered 6times, there were many mice having no tumor. For this reason,mono-mIL-12-Fc was administered 3 times, and then the number of immunecells that infiltrated the mouse tumor was analyzed.

FIGS. 22D-22F show the results of measuring the number of total immunecells (FIG. 22D), CD4⁺ T cells (FIG. 22E), and CD8⁺ T (FIG. 22F) cellsthat infiltrated the tumor in the mice sacrificed 3 days after the thirdadministration in FIGS. 21A-21E.

Specifically, after treatment as shown in FIGS. 21A-21E, the mouse tumorwas dissected 24 days after tumor transplantation and weighed. Then, thetumor was crushed using a wire mesh and collagenase (100 μg/ml) in aPetri dish and centrifuged in 10 ml of 2% FBS-containing medium at 50 gfor 5 minutes to remove the parenchymal tissue. Next, 1 ml of red bloodcell lysis buffer was added thereto to lyse red blood cells, and theresulting cells were washed with PBS to prepare a cell suspension, andthe number of the cells was countered with a hemocytometer. APC, FITC,or PE-cy5-conjugated anti-CD45, anti-CD3, anti-CD4, and anti-CD8antibodies were added to the cells isolated from tumor which were thenstained at 4° C. for 30 minutes, washed with cold PBS (pH 7.4), and thenanalyzed by flow cytometry (FACS Calibur,BD Bioscience) and Flow jo(Thermo Fisher Scientific). Each sample was analyzed by dot plots, andthen the CD45⁺ cell population (FIG. 22D), the CD45⁺CD3⁺CD4⁺ cellpopulation (FIG. 22E) and the CD45⁺CD3⁺CD8⁺ cell population (FIG. 22F)were defined as total tumor infiltrating immune cells, tumorinfiltrating CD4⁺ T cells and tumor infiltrating CD8⁺ T cells,respectively. The proportions of these cells relative to the cellsisolated from the whole tumor were calculated and multiplied by the cellnumber counted with a hemocytometer, and then the number of total tumorinfiltrating immune cells, tumor infiltrating CD4⁺ T cells and tumorinfiltrating CD8⁺ T cells that increased after administration ofmono-mIL-12-Fc was analyzed.

As a result, it could be seen that, in comparison with the control,bi-mIL-12-Fc and mono-mIL-12-Fc concentration-dependently increased thenumber of total immune cells, CD4⁺ T cells and CD8⁺ T cells thatinfiltrated the tumor. At the equimolar concentration, mono-mIL-12-Fcsignificantly increased the total immune cells (FIG. 22D), CD4⁺ T cells(FIG. 22E) and CD8⁺ T cells (FIG. 22F) that infiltrated the tumor,compared to bi-mIL-12-Fc. As a result, it was shown that mono-mIL-12-Fccaused greater infiltration of CD4+T cells (FIG. 22E) and CD8⁺ T cells(FIG. 22F) in tumor, accounting for the stronger tumor growthinhibition, compared to bi-mIL12-Fc.

Example 17 Evaluation of the Effects of Mono-mIL-12-Fc on CytokineSecretion from Immune Cells In Vivo and Increase in Cytotoxicity

IL-12 is known to inhibit the growth of cancer cells by increasing thesecretion of IFN-γ from T cells and NK cells (Trinchieri, 2003). Inaddition, IL-12 exhibits anticancer effects by enhancing the directcytotoxic effects of cytotoxic T cells and natural killer cells againstcancer cells. Thus, analysis was performed to determine whether the highanticancer effect of mono-IL-12-Fc would be attributable to an increasein the serum IFN-γ concentration of tumor-transplanted mice and to theenhancement of the direct cytotoxic effect of cytotoxic T cells andnatural killer cells against cancer cells.

FIGS. 23A-23B show the results of an ELISA performed to measure theconcentration of IFN-γ in the serum separated from the blood sampledfrom mouse facial veins at 24 hours after the last administration inFIGS. 21A-21E.

Specifically, at 24 hours after the last administration of the mIL-12-Fcfusion protein in FIGS. 20A-20B, blood was sampled from the facial veinsof the mice. The blood was allowed to stand at room temperature for 2hours so as to induce blood coagulation, and then centrifuged at 8000rpm for 10 minutes, and the supernatant serum was collected. In order tomeasure the concentration of IFN-γ in the serum, a 96-well plate (ThermoFisher Scientific) for ELISA was coated with mouse IFN-γ captureantibody for 12 hours, washed with PBST (PBS with 0.1% Tween-20), andthen blocked with 1% BSA (PBS with 1% bovine serum albumin) at roomtemperature for 1 hour. After washing with PBST (PBS with 0.1%Tween-20), the serum was diluted 10-fold with 1% BSA, and incubated atroom temperature for 2 hour. After washing with PBST (PBS with 0.1%Tween-20), each well was incubated with biotin-conjugated mouse IFN-γdetection antibody (Thermo Fisher Scientific) at room temperature for 1hour. After washing with PBST (PBS with 0.1% Tween-20), each well wasincubated with avidin-conjugated horseradish peroxidase (HRP) (ThermoFisher Scientific) at room temperature for 30 minutes, washed with PBST(PBS with 0.1% Tween-20), and then treated with3,3′,5,5′-tetramethylbenzidine substrate (TMB, sigma-aldrich). Theabsorbance at 450 nm was measured using a microplate reader. As shown inFIG. 23B, the serum IFN-γ concentration of the mice administered withbi-mIL-12-Fc did not increase compared to that of the control group.However, it was observed that the serum IFN-γ levels were increased inthe mice receiving the mono-mIL12-Fc treatment in proportion to the doseup to an equivalent molar amount of 1 mg rmIL12 compared to that of thecontrol group (FIG. 23A). In addition, it was shown that the tumorformation inhibitory effect of mono-mIL-12-Fc was because mono-mIL-12-Fcincreased the secretion of IFN-γ known to have the effect of inhibitingthe proliferation of some cancer cells.

In the tumor-transplanted mice treated with bi-mIL12-Fc, serum levels ofIFN-γ were low (FIG. 23B). Thus, in order to determine whetherbi-mIL-12-Fc had a low ability to induce IFN-γ secretion from NK cellsand T cells, the serum IFN-γ concentration was measured at indicatedtime points after single administration of mono-mIL-12-Fc andbi-mIL-12-Fc.

FIG. 23C shows the results of an ELISA performed to measure theconcentration of IFN-γ in serum at various indicated time points aftersingle intraperitoneal administration of bi-mIL-12-Fc and mono-mIL-12-Fcto Balb/c mice transplanted with CT26^(HERS/Neu) colorectal cancercells.

Specifically, when the tumor volume in the Balb/c mice transplanted withCT26^(HERS/Neu) colorectal cancer cells reached 300 mm³, bi-mIL-12-Fcand mono-mIL-12-Fc was administered intraperitoneally at a concentrationequimolar to 1 μg rmlL-12. After 1, 3 and 5 days, blood was sampled fromthe facial veins of the mice. The blood was allowed to stand at roomtemperature for 2 hours so as to induce blood coagulation andcentrifuged at 8000 rpm for 10 minutes, and the supernatant serum wascollected. In order to measure the concentration of IFN-γ in the serum,a 96-well plate (Thermo Fisher Scientific) for ELISA was coated withmouse IFN-γ capture antibody for 12 hours, washed with PBST (PBS with0.1% Tween-20), and then blocked with 1% BSA (PBS with 1% bovine serumalbumin) at room temperature for 1 hour. After washing with PBST (PBSwith 0.1% Tween-20), the serum was diluted 10-fold with 1% BSA, andincubated at room temperature for 2 hour. After washing with PB ST (PBSwith 0.1% Tween-20), each well was incubated with biotin-conjugatedmouse IFN-γ detection antibody (Thermo Fisher Scientific) at roomtemperature for 1 hour. After washing with PB ST (PBS with 0.1%Tween-20), each well was incubated with avidin-conjugated horseradishperoxidase (HRP) (Thermo Fisher Scientific) at room temperature for 30minutes, washed with PBST (PBS with 0.1% Tween-20), and then treatedwith 3,3′,5,5′-tetramethylbenzidine substrate (TMB, sigma-aldrich). Theabsorbance at 450 nm was measured using a microplate reader. As shown inFIG. 23C, in the tumor-transplanted mice, the group administered withbi-mIL-12-Fc showed a serum IFN-γ concentration similar to that of themono-mIL-12-Fc group up to 5 days, suggesting that bi-mIL-12-Fc has nointrinsic defect in the ability to induce IFN-γ secretion from effectorcells.

FIG. 23D is a graph showing the results of measuring the cytotoxiceffect of cytotoxic T cells, isolated from the spleen of mice sacrificed3 days after the last administration in FIGS. 21A-21E, against CT26^(HERS/Neu) cancer cells.

Specifically, 72 hours after the last administration of the cytokine inFIGS. 21A-21E, the mice were sacrificed, and the spleen was dissectedtherefrom and crushed in a 60 mm dish containing a 70-micron mesh andPBS. To the cells obtained by centrifugation, red blood cell lysisbuffer was added to lyse red blood cells. Next, the cells were washedwith PBS and incubated with APC-conjugated anti-CD3 antibody (ThermoFisher Scientific) and PE-conjugated anti-CD8 antibody at 4° C. for 30minutes. After the cells were washed with PBS, cytotoxic T cells(CD3⁺CD8⁺) were isolated using FACS Aria III (BD biosciences, Korea). Tomeasure the cytotoxic effect of the cytotoxic T cells against targetCT26 ^(HER2/Neu) cancer cells, the CT26^(HER2/Neu) cancer cells werestained with calcein AM (Thermo Fisher Scientific Inc., 10 μM).CT26^(HER2/Neu) cancer cells (2×10⁶) were suspended in 2 ml of DPBS, andmixed with 2μl of calcein AM (10 mM), and then incubated at 37° C. under5% CO₂ for 45 minutes. After washing with 10 ml of 10% FBS-containingRPMI1640, the cells were added to each well of a 96-well plate at adensity of 2×10⁴ cells per well, and cytotoxic T cells (1×10⁵/100μl/well) were added to each well and incubated at 37° C. under 5% CO₂for 4 hours. Living CT26^(HER2/Neu) cancer cells showing greenfluorescence and dead CT26^(HER2/Neu) cancer cells showing no greenfluorescence were analyzed by flow cytometry, and the cytotoxic effectof the cytotoxic T cells was expressed as percentage. It was shown thatthe cytotoxic T cells isolated from the tumor-transplanted miceadministered with mono-mIL-12-Fc showed a higher cytotoxic effectagainst target CT26^(HER2/Neu) cancer cells compared to the cytotoxic Tcells isolated from the tumor-transplanted mice administered withbi-mIL-12-Fc or the cytotoxic T cells isolated from the control group(FIG. 23D). In addition, it was shown that the tumor formationinhibitory effect of mono-mIL-12-Fc was attributed to the directcytotoxic effect of some cytotoxic T cells against cancer cells.

FIGS. 23E-23G show the results of measuring the cytotoxic effect ofcytotoxic T cells, isolated from the spleen of mice sacrificed 3 daysafter the third administration in FIGS. 21A-21E, using CT26^(HER2/Neu)cancer cells expressing tumor antigen and 4T1 cells expressing no tumorantigen, in order to determine whether the cytotoxic effect of cytotoxicT cells that was enhanced by administration of mono-IL-12-Fc to thetumor-transplanted mice would be tumor antigen-specific.

Specifically, 72 hours after the third administration of mono-IL-12-Fcin FIGS. 20A-20B, the mice were sacrificed, and the spleen was dissectedtherefrom and crushed in a 60-mm dish containing a 70-micron mesh andPBS. In order to measure the cytotoxic effect of cytotoxic T cellsagainst target CT26^(HERS/Neu) cancer cells (FIG. 23E) and non-target4T1 cells (FIG. 23F), the CT26^(HERS/Neu) cancer cells and the 4T1cancer cells were stained with calcein AM (Thermo Fisher ScientificInc., 10 μM) according to the method used for FIGS. 21M-21N. Afterwashing three times with 10 ml of 10% FBS-containing RPMI1640, the cellswere added to each well of a 96-well plate at a density of 2×10⁴ cellsper well, and cytotoxic T cells (1×10⁵/100 μl/well) were added to eachwell and incubated in a 37° C. incubator under 5% CO₂ for 4 hours.Living CT26^(HERS/Neu) cancer cells showing green fluorescence and deadCT26^(HERS/Neu) cancer cells showing no green fluorescence or 4T1 cancercells were analyzed by flow cytometry, and the cytotoxic effect of thecytotoxic T cells was expressed as percentage. As a result, it was shownthat the cytotoxic effect of cytotoxic T cells that was enhanced byadministration of mono-mIL-12-Fc was target cell-specific (FIG. 23G).

FIG. 23H shows the results of measuring the cytotoxic effect of naturalkiller cells, isolated from the spleen of mice sacrificed 3 days afterthe third administration in FIGS. 21A-21E, against CT26^(HERS/Neu)cancer cells.

Specifically, 3 days after the third administration of the cytokine inFIGS. 21A-21E, the mice were sacrificed, and the spleen was dissectedtherefrom and crushed in a 70-mm dish containing a 70-micron mesh andPBS. To the cells obtained by centrifugation, red blood cell lysisbuffer was added to lyse red blood cells. Next, the cells were washedwith PBS and incubated with APC-conjugated anti-CD3 antibody (ThermoFisher Scientific) and PE-conjugated anti-CD49b antibody at 4° C. for 30minutes. After the cells were washed with PBS, natural killer cells(CD3⁻CD49b⁺) were isolated using FACS Aria III (BD biosciences, Korea).To measure the cytotoxic effect of the natural killer cells againsttarget CT26^(HER2/Neu) cancer cells, the CT26^(HER2/Neu) cancer cellswere stained with calcein AM (Thermo Fisher Scientific Inc., 10 μM).CT26^(HER2/Neu) cancer cells (2×10⁶) were suspended in 2 ml of DPBS, andmixed with 2 μl of calcein AM (10 mM), and then incubated at 37° C.under 5% CO₂ for 45 minutes. After washing with 10 ml of 10%FBS-containing RPMI1640, the cells were added to each well of a 96-wellplate at a density of 2×10⁴ cells per well, and natural killer cells(1×10⁵/100 μl/well) were added to each well and incubated at 37° C.under 5% CO₂ for 4 hours. Living CT26^(HERS/Neu) cancer cells showinggreen fluorescence and dead CT26^(HERS/Neu) cancer cells showing nogreen fluorescence were analyzed by flow cytometry, and the cytotoxiceffect of the natural killer cells was expressed as percentage. It wasshown that the natural killer cells isolated from the tumor-transplantedmice administered with mono-mIL-12-Fc showed a higher cytotoxic effectagainst target CT26^(HERS/Neu) cancer cells compared to the naturalkiller cells isolated from the tumor-transplanted mice administered withbi-mIL-12-Fc or the cytotoxic T cells isolated from the control group.In addition, it was shown that the tumor formation inhibitory effect ofmono-mIL-12-Fc was attributed to the direct cytotoxic effect of somenatural killer cells against cancer cells.

Example 18 Evaluation of the Ability of mono-mIL-12-Fc to Form EffectorCD8⁺ T Cells and Memory CD8⁺ T Cells In Vivo

The production of adaptive immunity in tumor-transplanted mice isevaluated by whether effector memory CD8⁺ T cells and memory CD8⁺ Tcells are generated. Whether the tumor removal effect of mono-mIL-12-Fcwould be attributable to the generation of effector memory CD8⁺ T cellsand memory CD8⁺ T cells was measured.

FIGS. 24A, 24B and 24C show the results of measuring the number ofeffector CD8⁺ T cells (FIG. 24A), effector memory CD8⁺ T cells (FIG.24B) and memory CD8⁺ T cells (FIG. 24C) produced when mono-mIL-12-Fc wasadministered to tumor-bearing mice.

Specifically, after treatment as shown in FIGS. 21A-21E, the mousespleen was dissected 34 days after tumor transplantation, crushed usinga wide mesh in a Petri dish, and then washed with 10 ml of 2%FBS-containing medium. Next, 1 ml of red blood cell lysis buffer wasadded thereto to lyse red blood cells, and the resulting cells werewashed with PBS to prepare a spleen cell suspension, and the number ofthe cells was countered with a hemocytometer. APC, FITC, PE orPE-cy5-conjugated anti-CD3, anti-CD8, anti-CD62L, and anti-IL-7 receptor(IL-7R) antibodies were added to the spleen cells which were thenstained at 4° C. for 30 minutes, washed with cold PBS (pH 7.4), and thenanalyzed by flow cytometry (FACS Calibur, BD Bioscience) and Flow jo(Thermo Fisher Scientific). Each sample was analyzed by dot plots, andthe CD3⁺CD8⁺CD62L^(hi)IL-7R^(hi) cell population, theCD3⁺CD8⁺CD62L^(low)IL-7R^(hi) cell population and theCD3⁺CD8⁺CD62L^(hi)IL-7R^(hi) cell population were defined as effectorCD8⁺ T cells, effector memory CD8⁺ T cells and memory CD8⁺ T cells,respectively, and the proportions thereof relative to the total spleencells were calculated and multiplied by the cell number counted with ahemocytometer, and the number of effector CD8⁺ T cells, effector memoryCD8⁺ T cells and memory CD8⁺ T cells which increased afteradministration of mono-mIL-12-Fc was analyzed.

As a result, it could be seen that, in comparison with the control,mono-mIL-12-Fc concentration-dependently increased the number ofeffector memory CD8⁺ T cells and memory CD8⁺ T cells intumor-transplanted mice. However, bi-mIL-12-Fc increased the number ofeffector memory CD8⁺ T cells and memory CD8⁺ T cells only in the groupadministered at a concentration corresponding to an equivalent molaramount of 0.5 μg IL-12, and did not increase the number of effectormemory CD8⁺ T cells and memory CD8⁺ T cells in the group administeredwith the same at a concentration corresponding to an equivalent molaramount of 1μg IL-12. Thus, it was found that the higher tumor formationinhibitory effect of mono-mIL-12-Fc was attributed to the increasednumber of effector memory CD8⁺ T cells and memory CD8⁺ T cells, comparedto bi-mIL-12-Fc.

FIG. 24D shows the results obtained by re-transplanting CT26^(HERS/Neu)cancer cells into the survived mice 120 days after administration of 1μg mono-IL-12-Fc in FIGS. 21A-21E and measuring tumor volume changes inthe mice.

Specifically, 120 days after the last administration of 1 μgmono-IL-12-Fc to the female Balb/c mice (NARA Biotech, Korea) in FIGS.21A-21E, the survived mice were shaved, and CT26^(HER2/Neu) cells (1×10⁶cells/mouse) diluted in 150 μL of PBS were transplanted subcutaneouslyinto the mice. Next, the tumor was measured twice a week withoutadditional administration of 1 μg mono-IL-12-Fc, and the tumor volume(V) was calculated using the following equation: V=length×width/2. As aresult, it could be seen that, in comparison with the control group, thetumor in the mice that survived after administration of 1 μgmono-mIL-12-Fc started to decrease from 11 days. Thus, it was found thatwhen mono-mIL-12-Fc was administered to the tumor-transplanted mice, itproduced effector memory CD8⁺ T cells and memory CD8⁺ T cells, and thuseven when a tumor was transplanted again into the mice, it would beremoved.

Example 19 Evaluation of the Ability of mono-mIL-12-Fc to Form MemoryPrecursor Effector CD8⁺ T Cells In Vivo

In Examples 16 and 18, it was observed that the effect of bi-mIL-12-Fcon increasing the number of CD8⁺ T cells, effector memory CD8⁺ T cellsand central memory CD8⁺ T cells in tumor-transplanted mice was lowerthan that of mono-mIL-12-Fc. It was reported that after the effectorphase in which activated CD8⁺ T cells directly destroy tumor cells,effector CD8⁺ T cells partially differentiate into memory precursoreffector cells (MPECs) and then into memory CD8⁺ T cells, and mostlydifferentiate into short-lived effector cells (SLECs). Thus, analysiswas performed to determine whether CD8⁺ T cells activated byadministration of bi-mIL-12-Fc would differentiate into short-livedeffector cells, and thus the number of memory CD8⁺ T cells produced wassmall so that they could not remove tumors.

FIGS. 24E-24G show the results of analyzing the proportions of memoryprecursor effector cells (KLRG1⁻IL-7R⁺) and short-lived effector cells(KLRG1⁺IL-7R⁻) in the CD8⁺ T cells present in the spleen of micesacrificed 3 days after the third administration in FIGS. 21A-21E. FIG.24E are flow cytometry dot plots showing the proportion of memoryprecursor effector cells (KLRG1⁻IL-7R⁺) and short-lived effector cells(KLRG1⁺IL-7R⁻) among CD8⁺ T cells in the spleen isolated fromtumor-bearing mice sacrificed 3 days after the third administration inFIGS. 21A-21E. FIGS. 24F-24G are graphs showing the results of flowcytometry performed to analyze the proportion of memory precursoreffector cells (KLRG1⁻IL-7R⁺) (FIG. 24F) and short-lived effector cells(KLRG1⁺IL-7R⁻) (FIG. 24G) among CD8⁺ T cells in the spleen isolated fromtumor-bearing mice sacrificed 3 days after the third administration inFIGS. 21A-21E.

Specifically, after treatment as shown in FIGS. 21A-21E, the mousespleen was dissected 24 days after tumor transplantation, crushed usinga wide mesh in a Petri dish, and then washed with 10 ml of 2%FBS-containing medium. Next, 1 ml of red blood cell lysis buffer wasadded thereto to lyse red blood cells, and the resulting cells werewashed with PBS to prepare a cell suspension. APC, FITC, PE orPE-cy5-conjugated anti-CD3, anti-CD8, anti-KLRG1, and anti-IL-7 receptor(IL-7R) antibodies were added to the spleen cells which were thenstained at 4° C. for 30 minutes, washed with cold PBS (pH 7.4), and thenanalyzed by flow cytometry (FACS Calibur, BD Bioscience) and Flow jo(Thermo Fisher Scientific). Each sample was analyzed by dot plots, andthe CD3⁺CD8⁺KLRG1⁻IL-7R⁺ cell population and the CD3⁺CD8⁺KLRG1⁺IL-7R⁻cell population were defined as memory precursor effector cells andshort-lived effector cells, respectively, and the proportions thereofrelative to the total spleen cells were analyzed.

As a result, it could be seen that, in comparison with the control,mono-mIL-12-Fc concentration-dependently increased the proportion ofmemory precursor effector cells in the tumor-transplanted mice. However,administration of bi-mIL-12-Fc did not increase the proportion of memoryprecursor effector cells compared to control, but rather increase thenumber of short-lived effector cells. Thus, it was found thatmono-mIL-12-Fc significantly increased the number of effector memoryCD8⁺ T cells and memory CD8⁺ T cells, compared to bi-mIL-12-Fc bypromoting production of memory precursor effector cells, indicating thatit has a higher effect on tumor removal.

Example 20 Evaluation of the Effect of mono-mIL-12-Fc on Expression ofTranscription Factors Involved in Induction of Memory CellDifferentiation

It was reported that when CD8⁺ T cells were administered with highconcentrations of IL-12 or were activated by administering IL-12frequently for 2 days or more, expression of the transcription factorT-bet that allows CD8⁺ T cells to differentiate into short-livedeffector cells increases and expression of the transcription factoreomesodermin (Eomes) that allows CD8⁺ T cells to differentiate intomemory precursor effector cells decreases. Thus, analysis was performedto determine whether mono-mIL-12-Fc and bi-mIL-12-Fc woulddifferentially regulate the expression of T-bet and Eomes in CD8⁺ Tcells so as to change the proportion of CD8⁺ T cells that differentiateinto short-lived effector cells.

FIGS. 25A and 25B show the results of flow cytometry analysis performedto measure the proportions of CD8⁺ T cells (which show high expressionof T-bet (FIG. 25A) that inhibits memory cell differentiation) and CD8⁺T cells (which show low expression of Eomes (FIG. 25B) that promotesmemory cell differentiation) in the spleen of mice sacrificed 3 daysafter the third administration in FIGS. 21A-21E.

Specifically, after treatment as shown in FIGS. 21A-21E, the mousespleen was dissected 24 days after tumor transplantation, crushed usinga wire mesh in a Petri dish, and then washed with 10 ml of 2%FBS-containing medium. Next, 1 ml of red blood cell lysis buffer wasadded thereto to lyse red blood cells, and the resulting cells werewashed with PBS to prepare a cell suspension. The spleen cells werestained with PE-cy5- or FITC-conjugated anti-CD3 and anti-CD8 antibodiesat 4° C. for 30 minutes and washed with cold PBS (pH 7.4). Then, thecells were fixed with Foxp3/Transcription Factor Staining Buffer Set(Thermo Fisher Scientific) (which is an intranuclear transcriptionfactor staining reagent), and permeabilized. Next, the cells werestained with PE- or efluor 660-conjugated anti-T-bet or anti-Eomesantibody at 4° C. for 30 minutes, and then analyzed by flow cytometry(FACS Calibur, BD Bioscience) in permeabilization buffer and Flow jo(Thermo Fisher Scientific) for flow cytometry data analysis. Each samplewas analyzed by dot plots, and the proportions of theCD3⁺CD8⁺T-bet^(high) cell population (FIG. 25A) and theCD3⁺CD8⁺Eomes+T-bet^(low) cell population (FIG. 25B) were analyzed. As aresult, it could be seen that, in comparison with the control,mono-mIL-12-Fc concentration-dependently reduced the proportion of theCD3⁺CD8⁺T-bet^(high) cell population and increased the proportion of theCD3⁺CD8⁺ Eomes+T-bet^(low) cell population. However, bi-mIL-12-Fcreduced the proportion of the CD3⁺CD8⁺T-bet^(h)ig^(h) cell populationonly in the group administered with the same at a concentrationcorresponding to an equivalent molar amount of 0.5 μg IL-12 andincreased the proportion of the CD3⁺CD8⁺Eomes⁺T-bet^(low) cellpopulation in the group. In addition, in the group administered withbi-mIL-12-Fc at a concentration corresponding to an equivalent molaramount of 1μg IL-12, bi-mIL-12-Fc did not show the effect of reducingthe proportion of the CD3⁺CD8⁺T-bet^(high) cell population or increasingthe proportion of the CD3⁺CD8⁺Eomes⁺T-bet^(low) cell population. Thus,it was found that, in comparison with bi-mIL-12-Fc, mono-mIL-12-Fc had ahigher effect of removing tumors by reducing the proportion of theCD3⁺CD8⁺T-bet^(high) cell population and increasing the proportion ofthe CD3⁺CD8⁺Eomes⁺T-bet^(low) cell population so as to significantlyincrease the number of effector memory CD8⁺ T cells and memory CD8⁺ Tcells.

It is known that when CD8⁺ T cells are stimulated with inflammatorycytokines such as IL-12 in the presence of a T cell receptor signal anda co-stimulatory signal, the phosphorylation of STAT4 increases and thephosphorylated STAT4 (pSTAT4) migrates into the nucleus and binds to theT-bet enhancer, thereby increasing the expression of T-bet. Thus,analysis was performed to determine whether the differentiation of CD8⁺T cells into short-lived effector cells, which occurred whenbi-mIL-12-Fc was administered at a concentration corresponding to anequivalent molar amount of 1μg IL-12, would be because administration ofbi-mIL-12-Fc at a concentration corresponding to an equivalent molaramount of 1μg IL-12 increased the expression of pSTAT4 and T-bet when Tcells were activated in the tumor draining lymph nodes of thetumor-transplanted mice, compared to mono-mIL-12-Fc.

FIG. 25C shows the results of flow cytometry analysis performed tomeasure the expression level of phosphorylated STAT4 in CD8⁺ T cellsisolated from the tumor draining lymph node on 24 hours afterintraperitoneally administrating bi-mIL-12-Fc and mono-mIL-12-Fc once ata concentration corresponding to equivalent molar amount of 1μg rmIL-12when the tumor volume in the B alb/c mice transplanted withCT26^(HERS/Neu) reached 300 mm³.

Specifically, as described with respect to FIG. 23C, when the tumorvolume in the Balb/c mice transplanted with CT26^(HERS/Neu) colorectalcancer cells reached 300 mm³, bi-mIL-12-Fc and mono-mIL-12-Fc wereadministered intraperitoneally into the mice at a concentrationequimolar to 1 μg rmIL-12. After 24 hours, the tumor draining lymph nodeof the mice was dissected, crushed using a wire mesh in a Petri dish,and then washed with 10 ml of 2% FBS-containing medium. Next, 1 ml ofred blood cell lysis buffer was added thereto to lyse red blood cells,and the resulting cells were washed with PBS, thus preparing a cellsuspension. The draining lymph node cells were stained with PE-cy5- orFITC-conjugated anti-CD3 and anti-CD8 antibodies at 4° C. for 30minutes, washed with PBS (pH 7.4), and then fixed in cold methanol.Next, the draining lymph node cells were washed with cold PBS (pH 7.4),stained with APC-conjugated anti-pSTAT4 antibody at 4° C. for 30minutes, washed with cold PBS (pH 7.4), and then analyzed by flowcytometry (FACS Calibur, BD Bioscience) and Flow jo (Thermo FisherScientific). Each sample was analyzed by dot plots, and the expressionlevels of pSTAT4 in CD3⁺CD8⁺T cells were compared. As a result, incomparison with mono-mIL-12-Fc, bi-mIL-12-Fc showed the effect ofincreasing the expression of pSTAT4 when CD8⁺T cells were activated inthe tumor draining lymph nodes of the tumor-transplanted mice.

FIG. 25D shows the results of flow cytometry performed to measure theproportion of CD8⁺ T cells (which express T-bet that inhibits memorycell differentiation) in the tumor draining lymph node on 72 hours aftersingle intraperitoneal administration in FIG. 25C.

Specifically, as described with respect to FIG. 23C, when the tumorvolume in the Balb/c mice transplanted with CT26^(HERS/Neu) colorectalcancer cells reached 300 mm³, bi-mIL-12-Fc and mono-mIL-12-Fc wereadministered intraperitoneally into the mice at a concentrationcorresponding to equivalent molar amount of 1 μg rmIL-12. After 72hours, the tumor draining lymph node of the mice was dissected, crushedusing a wire mesh in a Petri dish, and then washed with 10 ml of 2%FBS-containing medium. Next, 1 ml of red blood cell lysis buffer wasadded thereto to lyse red blood cells, and the resulting cells werewashed with PBS, thus preparing a cell suspension. The draining lymphnode cells were stained with PE-cy5- or FITC-conjugated anti-CD3 andanti-CD8 antibodies at 4° C. for 30 minutes, washed with PBS (pH 7.4),fixed using Foxp3/Transcription Factor Staining Buffer Set (ThermoFisher Scientific) (which is an intranuclear transcription factorstaining reagent), and then permeabilized. Next, the cells were stainedwith PE- or APC-conjugated anti-T-bet antibody at 4° C. for 30 minutes,and then analyzed by flow cytometry (FACS Calibur (BD Bioscience) inpermeabilization buffer and Flow jo (Thermo Fisher Scientific) analysis.Each sample was analyzed by dot plots, and the proportion of CD3⁺CD8⁺ Tcells expressing T-bet was compared. As a result, in comparison withmono-mIL-12-Fc, bi-mIL-12-Fc showed the effect of increasing theexpression of T-bet when CD8⁺T cells were activated in the draininglymph lodes of the tumor-transplanted mice. Thus, it was found that thedifferentiation of CD8⁺ T cells into short-lived effector cells, whichoccurred when bi-mIL-12-Fc was administered at a concentrationcorresponding to equivalent molar amount 1 μg IL-12, was becauseadministration of bi-mIL-12-Fc increased the expression of pSTAT4 andT-bet, compared to mono-mIL-12-Fc, when T cells in the tumor draininglymph nodes of the tumor-transplanted mice were activated.

FIGS. 25E-25H show the result of measuring whether when mono-mIL-12-Fcwas cross-reacted with anti-Fc antibody, like bi-mIL-12-Fc expressingtwo L-12 molecules, so that CD8⁺ T cells could be stimulated by two L-12molecules, the expression of pSTAT4 and T-bet in the cells would beincreased to a level similar to the level shown when the cells weretreated with bi-mIL-12-Fc.

Specifically, spleens and tumor draining lymph nodes were dissected fromnormal Balb/c mice, crushed using a wire mesh in a Petri dish, and thenwashed with 10 ml of 2% FBS-containing medium. Next, 1 ml of red bloodcell lysis buffer was added thereto to lyse red blood cells, and theresulting cells were washed with PBS, thus preparing a cell suspension.The lymph node cells were stained with PE-conjugated anti-CD8 antibodyat 4° C. for 30 minutes, washed with cold PBS (pH 7.4), and incubatedwith anti-PE microbeads (Miltenyi Biotec) for 15 minutes, and CD8⁺ Tcells were separated therefrom using a MACS separator and an LS column(Miltenyi Biotec). 100 ∞l of 0.5 μg/ml of anti-CD3 antibody was added toeach well of a 96-well round bottom plate which was then incubated at 4°C. for 12 hours and washed with PBS to remove anti-CD3 antibody notattached to the plate, and 50 μl of 2 μg/ml of anti-CD28 antibody wasadded to each well. Next, mono-mIL-12-Fc and bi-mIL-12-Fc were reactedwith various concentrations of anti-Fc antibody at 4° C. for 30 minutes,and then added to each well at a concentration equimolar to 20 μM IL-12.Next, CD8⁺ T cells (4×10⁴/well) were added to each well and incubated ina 37° C. incubator for 3 hours in order to measure the expression ofpSTAT4 and for 3 days in order to measure the expression of T-bet. Tomeasure the expression of pSTAT4 and T-bet, the cells were stainedaccording to the method described with respect to FIGS. 25C and 25D, andwere then analyzed by flow cytometry. Each sample was analyzed by dotplots, and the expression levels of pSTAT4 (FIG. 25E and FIG. 25F) orT-bet in the CD8⁺ T cells (FIG. 25G and FIG. 25H) were compared. As aresult, it was shown that when mono-mIL-12-Fc was cross-reacted withanti-Fc antibody so that CD8⁺ T cells could be stimulated by two IL-12molecules, the expression levels of pSTAT4 and T-bet in the cellsincreased to the levels shown when the cells were treated withbi-mIL-12-Fc.

In conclusion, as shown in FIG. 26, in comparison with bi-mIL-12-Fc,mono-mIL-12-Fc induces low expression of pSTAT4 and T-bet in CD8⁺ Tcells so that the CD8⁺ T cells can differentiate into memory precursoreffector cells and then into effector memory cells and central memorycells. Thus, mono-mIL-12-Fc can remove tumors from tumor-transplantedmice even at low concentration (corresponding to equivalent molar amount0.5 μg IL-12), thus prolonging the life-span of the mice. However,bi-mIL-12-Fc induces high expression of pSTAT4 and T-bet in CD8⁺ T cellsso that the cells can differentiate into short-livered effector cellsprecluding the development of memory cells. Thus, when bi-mIL-12-Fc isadministered at the same molar concentration as that of mono-mIL-12-Fc,it cannot completely remove tumors from tumor-transplanted mice. Thus,only when bi-mIL-12-Fc is administered at higher concentration(corresponding to equivalent molar amount of 2 μg IL-12) and cytotoxicCD8⁺ T cells are expanded in the effector phase that directly destroystumor cells, bi-mIL-12-Fc can remove tumors.

INDUSTRIAL APPLICABILITY

The heterodimeric Fc-fused protein according to the present inventionhas an advantage in that it can retain the activity of a naturallyoccurring physiologically active protein, which is composed of two ormore different subunit proteins and thereby exhibit the physiologicalactivity by forming an assembled protein, because each subunit of theprotein can be separately fused to each chain of heterodimeric Fc ofimmunoglobulin such that the fused protein can maintain the naturallyoccurring form and structure to the highest possible degree. Inaddition, the in vivo half-life of the physiologically active proteincontained in the heterodimeric Fc-fused protein can be significantlyincreased due to the heterodimeric Fc-mediated long half-life such thatthe physiological activities thereof in vivo can be long-lasting.

Further, the heterodimeric Fc-fused protein according to the presentinvention has an advantage in that it is possible to easily produce aheterodimeric Fc-fused protein in the native configuration without needto optimize an additional purification process.

Although the present invention has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only for a preferred embodiment anddoes not limit the scope of the present invention. Thus, the substantialscope of the present invention will be defined by the appended claimsand equivalents thereof.

1.-17. (canceled)
 18. A method for treating cancer, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of a pharmaceutical composition comprising aheterodimeric Fc-fused protein comprising a first Fc region and a secondFc region of an immunoglobulin Fc (fragment crystallizable) pair and thep40 and p35 subunits of IL-12, wherein the p40 and p35 subunits are eachlinked to the N-terminus or C-terminus of the Fc regions, and whereinCH3 domains of the first Fc region and the second Fc region eachcomprise one or more mutations promoting heterodimerization.
 19. Themethod of claim 18, wherein the p40 and p35 subunits are each linked tothe N-terminus of the Fc regions.
 20. The method according to claim 19,wherein the p40 or p35 subunit is linked to its corresponding Fc regionby a linker.
 21. The method according to claim 20, wherein the p35subunit is linked to its corresponding Fc region by a linker.
 22. Themethod according to claim 21, wherein the linker comprises a (G₄S)₃linker.
 23. The method according to claim 18, wherein each of the firstFc region and the second Fc region is from an Fc region selected fromthe group consisting of human IgG1, IgG2, IgG3, and IgG4.
 24. The methodaccording to claim 18, wherein the first Fc region and the second Fcregion are included in a whole antibody form selected from the groupconsisting of human IgG1, IgG2, IgG3, and IgG4.
 25. The method accordingto claim 18, wherein CH3 domains of the first Fc region and the secondFc region each comprise one or more mutations, wherein the mutation atthe CH3 domain of the first Fc region or the second Fc region includesone or more mutations selected from the group consisting of: (a)glutamic acid (E), arginine (R), methionine (M), aspartic acid (D), orhistidine (H) substitution of the amino acid residue at position 370 inthe CH3 domain of the first Fc region; (b) glutamic acid (E)substitution of the amino acid residue at position 360 in the CH3 domainof the first Fc region; (c) tryptophan (W) substitution of the aminoacid residue at position 409 in the CH3 domain of the first Fc region;(d) asparagine (N), aspartic acid (D), alanine (A), isoleucine (I),glycine (G), or methionine (M) substitution of the amino acid residue atposition 357 in the CH3 domain of the second Fc region, and threonine(T) or tryptophan (W) substitution of the amino acid residue at position364 in the CH3 domain of the second Fc region; and (e) threonine (T)substitution of the amino acid residue at position 405 in the CH3 domainof the second Fc region, and valine (V) substitution of the amino acidresidue at position 399 in the CH3 domain of the second Fc region,wherein mutation positions are numbered according to the EU index. 26.The method according to claim 25, wherein the CH3 domain of the secondFc region further comprises: arginine (R) substitution of the amino acidresidue at position 347, wherein the positions are numbered according tothe EU index.
 27. The method according to claim 26, wherein the CH3domains in the first Fc region and the second Fc region further includethe following residues: (i) cysteine (C) substitution at position 349 inthe CH3 domain of the first Fc region; and (ii) cysteine (C)substitution at position 354 in the CH3 domain of the second Fc region,wherein the positions are numbered according to the EU index.
 28. Themethod according to claim 18, wherein the IL-12 is human IL-12.
 29. Themethod according to claim 18, wherein the cancer is selected from thegroup consisting of colorectal cancer, melanoma, breast cancer,pancreatic cancer, kidney cancer, prostate cancer, ovarian cancer, smallintestine cancer, esophageal cancer, cervical cancer, lung cancer,lymphoma, and blood cancer.
 30. The method according to claim 18,wherein the pharmaceutical composition is used in combination withanother anticancer therapy.